Use of sulfonamide inhibitors of bcl-2 and bcl-xl to treat ophthalmic disease by selectively removing senescent cells

ABSTRACT

Methods are provided herein for selectively killing senescent cells and for treating senescence-associated diseases and disorders by administering a senolytic agent. Senescence-associated diseases and disorders treatable by the methods using the senolytic agents described herein include cardiovascular diseases and disorders associated with or caused by arteriosclerosis, such as atherosclerosis; idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease; osteoarthritis; senescence-associated ophthalmic diseases and disorders; and senescence-associated dermatological diseases and disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/955,542, filed Apr. 17, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/647,688, filed Jul. 12, 2017, now U.S. Pat. No.9,980,962, and is a continuation of U.S. patent application Ser. No.15/827,539, filed Nov. 30, 2017, now U.S. Pat. No. 10,010,546, which isa continuation of U.S. application Ser. No. 15/455,575, filed Mar. 10,2017, now U.S. Pat. No. 9,849,128, which is a continuation of U.S.patent application Ser. No. 15/114,762, filed Jul. 27, 2016, now U.S.Pat. No. 9,993,472, which is the U.S. National Stage ofPCT/US2015/013387, filed Jan. 28, 2015; which claims the prioritybenefit of U.S. Provisional Application 62/061,629, filed Oct. 8, 2014,U.S. Provisional Application 62/061,627, filed Oct. 8, 2014, U.S.Provisional Application 62/057,828, filed Sep. 30, 2014, U.S.Provisional Application 62/057,825, filed Sep. 30, 2014, U.S.Provisional Application 62/057,820, filed Sep. 30, 2014, U.S.Provisional Application 62/044,664, filed Sep. 2, 2014, U.S. ProvisionalApplication 62/042,708, filed Aug. 27, 2014, U.S. ProvisionalApplication 62/002,709, filed May 23, 2014, U.S. Provisional Application61/979,911, filed Apr. 15, 2014, U.S. Provisional Application61/932,711, filed Jan. 28, 2014, U.S. Provisional Application61/932,704, filed Jan. 28, 2014. U.S. application Ser. No. 15/114,762and PCT/US2015/013387 are hereby incorporated herein by reference intheir entirety for all purposes.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under AG009909,AG017242, AG041122 and AG046061 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 44237-721-311_SEQUENCE_LISTING.txt. The textfile is 8 KB, was created on Aug. 3, 2018, and is being submittedelectronically via EFS-Web.

BACKGROUND Technical Field

The disclosure herein relates generally to methods for treatment andprophylaxis of senescent cell-associated diseases and disorders.

Description of the Related Art

Senescent cells accumulate in tissues and organs of individuals as theyage and are found at sites of age-related pathologies. Senescent cellsare believed important to inhibiting proliferation of dysfunctional ordamaged cells and particularly to constraining development of malignancy(see, e.g., Campisi, Curr. Opin. Genet. Dev. 21:107-12 (2011); Campisi,Trends Cell Biol. 11:S27-31 (2001); Prieur et al., Curr. Opin. CellBiol. 20:150-55 (2008)); nevertheless, the presence of senescent cellsin an individual may contribute to aging and aging-related dysfunction(see, e.g., Campisi, Cell 120:513-22 (2005)). Given that senescent cellshave been causally implicated in certain aspects of age-related declinein health and may contribute to certain diseases, and are also inducedas a result of necessary life-preserving chemotherapeutic and radiationtreatments, the presence of senescent cells may have deleterious effectsto millions of patients worldwide. However, identifying and developingtreatments of such diseases and conditions by selective elimination ofsenescent cells has been an arduous undertaking. The present disclosureaddresses these needs and offers related advantages.

BRIEF SUMMARY

Provided herein are methods for treating senescence-associated diseasesby administering a senolytic agent. The following are certainembodiments described in greater detail herein. As described herein thesenolytic agent is administered for a time sufficient and in an amountsufficient that selectively kills senescent cells. Also provided hereinare methods for selectively killing senescent cells in a subject who hasa senescence associated disease or disorder, which in certainembodiments is not a cancer, and which senolytic agents described hereinare administered to the subject in need thereof according to theadministration methods described herein.

In one embodiment, a method is provided for treating asenescence-associated disease or disorder comprising administering to asubject in need thereof a therapeutically-effective amount of a smallmolecule senolytic agent that selectively kills senescent cells overnon-senescent cells; wherein the senescence-associated disease ordisorder is not a cancer, wherein the senolytic agent is administered inat least two treatment cycles, wherein each treatment cycleindependently comprises a treatment course of from 1 day to 3 monthsfollowed by a non-treatment interval of at least 2 weeks; provided thatif the senolytic agent is an MDM2 inhibitor, the MDM2 inhibitor isadministered as a monotherapy, and each treatment course is at least 5days long during which the MDM2 inhibitor is administered on at least 5days. In certain embodiments, the senolytic agent is selected from anMDM2 inhibitor; an inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members wherein the inhibitor inhibits at least BCL-xL; and anAkt specific inhibitor. In a specific embodiment, the MDM2 inhibitor isa cis-imidazoline compound, a spiro-oxindole compound, or abenzodiazepine compound. In a specific embodiment, the cis-imidazolinecompound is a nutlin compound. In a specific embodiment, the senolyticagent is an MDM2 inhibitor and is Nutlin-3a or RG-1172. In a specificembodiment, the nutlin compound is Nutlin-3a. In a specific embodiment,the cis-imidazoline compound is RG-7112, RG7388, RO5503781, or is adihydroimidazothiazole compound. In a specific embodiment, thedihydroimidazothiazole compound is DS-3032b. In a specific embodiment,the MDM2 inhibitor is a spiro-oxindole compound selected from MI-63,MI-126, MI-122, MI-142, MI-147, MI-18, MI-219, MI-220, MI-221, MI-773,and3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one.In a specific embodiment, the MDM2 inhibitor is Serdemetan; apiperidinone compound; CGM097; or an MDM2 inhibitor that also inhibitsMDMX and which is selected from RO-2443 and RO-5963. In a specificembodiment, the piperidinone compound is AM-8553. In a specificembodiment, the inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members is a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-winhibitor; or a BCL-xL selective inhibitor. In a specific embodiment,the senolytic agent is an inhibitor of one or more BCL-2 anti-apoptoticprotein family members wherein the inhibitor inhibits at least Bcl-xLand is selected from ABT-263, ABT-737, WEHI-539, and A-1155463. In aspecific embodiment, the BCL-xL selective inhibitor is abenzothiazole-hydrazone compound, an aminopyridine compound, abenzimidazole compound, a tetrahydroquinolin compound, or a phenoxylcompound. In a specific embodiment, the benzothiazole-hydrazone compoundis a WEHI-539. In a specific embodiment, the inhibitor of the one ormore BCL-2 anti-apoptotic protein family members is A-1155463, ABT-263,or ABT-737. In a specific embodiment, the Akt inhibitor is MK-2206. In aspecific embodiment, the senolytic agent is an MDM2 inhibitor or is aninhibitor of one or more BCL-2 anti-apoptotic protein family memberswherein the inhibitor inhibits at least BCL-xL and is cytotoxic tocancer cells, the total dose of the senolytic agent administered duringeach treatment cycle is an amount ineffective for treating a cancer. Ina specific embodiment, the senolytic agent is an MDM2 inhibitor or is aninhibitor of one or more BCL-2 anti-apoptotic protein family memberswherein the inhibitor inhibits at least BCL-xL and is cytotoxic tocancer cells and wherein the senolytic agent is administered in two ormore treatment cycles, the total dose of the senolytic agentadministered during the two or more treatment cycles is an amount lessthan the amount effective for a cancer treatment. In a specificembodiment, the MDM2 inhibitor is Nutlin-3a; RG-7112; RG7388; RO5503781;DS-3032b; MI-63; MI-126; MI-122; MI-142; MI-147; MI-18; MI-219; MI-220;MI-221; MI-773; and3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one;Serdemetan; AM-8553; CGM097; or an MDM2 inhibitor that also inhibitsMDMX and which is selected from RO-2443 and RO-5963. In a specificembodiment, the inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members is ABT-263, ABT-737, A-1155463, or WEHI-539. In anotherembodiment, the subject has a cancer and wherein thesenescence-associated disease or disorder is a chemotherapy side effector radiotherapy side effect, wherein the senolytic agent is administeredto the subject on one or more days beginning on at least the sixth daysubsequent to an administration cycle of the chemotherapy orradiotherapy and not concurrent with the chemotherapy or radiotherapy,and wherein the senolytic agent is not a chemotherapeutic agent fortreating the cancer, and wherein the senolytic agent is a small moleculeand is selected from an MDM2 inhibitor; an inhibitor of one or moreBCL-2 anti-apoptotic protein family members wherein the inhibitorinhibits at least BCL-xL selected from a BCL-2/BCL-xL inhibitor; aBCL-2/BCL-xL/BCL-w inhibitor; and a BCL-xL selective inhibitor; and anAkt specific inhibitor. In another specific embodiment, thechemotherapeutic side effect is selected from gastrointestinal toxicity,peripheral neuropathy, fatigue, malaise, low physical activity,hematological toxicity, hepatotoxicity, alopecia, pain, mucositis, fluidretention, and dermatological toxicity. In another specific embodiment,the chemotherapeutic side effect is fatigue. In another specificembodiment, the chemotherapeutic side effect comprises cardiotoxicity.In another specific embodiment, the senescence-associated disease ordisorder is osteoarthritis, atherosclerosis, chronic obstructivepulmonary disease, or idiopathic pulmonary fibrosis. In another specificembodiment, administration of the senolytic agent comprises three ormore treatment cycles. In another specific embodiment, the senolyticagent is administered on one day, two days, three days, or four dayswith the proviso that the senolytic agent is not the MDM2 inhibitor. Inanother specific embodiment, the senolytic agent is administered as amonotherapy.

In another embodiment, a method is provided for treating asenescence-associated disease or disorder that is not a cancer,comprising administering to a subject in need thereof atherapeutically-effective amount of a small molecule senolytic agentthat selectively kills senescent cells over non-senescent cells andwhich agent is cytotoxic to cancer cells, wherein the senolytic agent isadministered as a monotherapy within at least one treatment cycle, whichtreatment cycle comprises a treatment course followed by a non-treatmentinterval; and wherein the total dose of the senolytic agent administeredduring the treatment cycle is an amount less than the amount effectivefor a cancer treatment, wherein the senolytic agent is (a) an inhibitorof a Bcl-2 anti-apoptotic protein family member that inhibits at leastBcl-xL; (b) an MDM2 inhibitor; or (c) an Akt specific inhibitor. Incertain embodiments, the senolytic agent is administered during two ormore treatment cycles, and wherein the total dose of the senolytic agentadministered during the two or more treatment cycles is an amount lessthan the amount effective for a cancer treatment.

In other specific embodiments of the methods described above and herein,each treatment course is no longer than (a) one month, or (b) no longerthan two months, or (c) no longer than 3 months. In a specificembodiment, each treatment course is no longer than (a) 5 days, (b) 7days, (c) 10 days, (d) 14 days, or (e) 21 days. In a specificembodiment, the senolytic agent is administered every 2^(nd) day orevery 3^(rd) day of each treatment course. In a specific embodiment, thetreatment course is one day, two days, three days, or four days. Inanother specific embodiment, the senolytic agent is administered dailyduring each treatment course. In another specific embodiment, thenon-treatment interval is at least two weeks, at least one month, atleast 2 months, at least 3 months, at least 6 months, at least 9 months,or at least 1 year. In another specific embodiment, the treatment courseis one In another specific embodiment, the senescence-associated diseaseor disorder is a cardiovascular disease selected from atherosclerosis,angina, arrhythmia, cardiomyopathy, congestive heart failure, coronaryartery disease, carotid artery disease, endocarditis, coronarythrombosis, myocardial infarction, hypertension, aortic aneurysm,cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia,mitral valve prolapsed, peripheral vascular disease, cardiac stressresistance, cardiac fibrosis, brain aneurysm, and stroke. In anotherspecific embodiment, the senescence-associated disease or disorder is aninflammatory or autoimmune disease or disorder selected fromosteoarthritis, osteoporosis, oral mucositis, inflammatory boweldisease, kyphosis, and herniated intervertebral disc. In anotherspecific embodiment, the senescence-associated disease or disorder is aneurodegenerative disease selected from Alzheimer's disease, Parkinson'sdisease, Huntington's disease, dementia, mild cognitive impairment, andmotor neuron dysfunction. In another specific embodiment, thesenescence-associated disease or disorder is a metabolic diseaseselected from diabetes, diabetic ulcer, metabolic syndrome, and obesity.In another specific embodiment, the senescence-associated disease ordisorder is a pulmonary disease selected from pulmonary fibrosis,chronic obstructive pulmonary disease, asthma, cystic fibrosis,emphysema, bronchiectasis, and age-related loss of pulmonary function.In another specific embodiment, the senescence-associated disease ordisorder is an eye disease or disorder selected from maculardegeneration, glaucoma, cataracts, presbyopia, and vision loss. Inanother specific embodiment, the senescence-associated disease ordisorder is an age-related disorder selected from renal disease, renalfailure, frailty, hearing loss, muscle fatigue, skin conditions, skinwound healing, liver fibrosis, pancreatic fibrosis, oral submucosafibrosis, and sarcopenia. In another specific embodiment, thesenescence-associated disease or disorder is a dermatological disease ordisorder is selected from eczema, psoriasis, hyperpigmentation, nevi,rashes, atopic dermatitis, urticaria, diseases and disorders related tophotosensitivity or photoaging, rhytides; pruritis; dysesthesia;eczematous eruptions; eosinophilic dermatosis; reactive neutrophilicdermatosis; pemphigus; pemphigoid; immunobullous dermatosis;fibrohistocytic proliferations of skin; cutaneous lymphomas; andcutaneous lupus. In another specific embodiment, thesenescence-associated disease or disorder is atherosclerosis;osteoarthritis; pulmonary fibrosis; hypertension, or chronic obstructivepulmonary disease. In another specific embodiment, the senolytic agentis administered directly to an organ or tissue that comprises thesenolytic cells. In another specific embodiment, the senolytic agent iscombined with at least one pharmaceutically acceptable excipient toformulate a pharmaceutically acceptable composition to providetimed-release of the senolytic agent. In another specific embodiment,the senolytic agent is administered as a bolus infusion. In anotherspecific embodiment, the senescence-associated disease or disorder isosteoarthritis and the senolytic agent is administered directly into theosteoarthritic joint. In another specific embodiment, the senolyticagent is administered intra-articularly to the osteoarthritic joint. Inanother specific embodiment, the senolytic agent is administeredtopically, transdermally, or intradermally. In another specificembodiment, the senescence-associated disease or disorder isosteoarthritis and the senolytic agent induces production of Type IIcollagen in a joint. In another specific embodiment, thesenescence-associated disease or disorder is osteoarthritis and thesenolytic agent inhibits erosion of a proteoglycan layer in a joint. Inanother specific embodiment, the senescence-associated disease ordisorder is osteoarthritis and the senolytic agent inhibits erosion of abone of a joint. In another specific embodiment, pulmonary fibrosis isidiopathic pulmonary fibrosis. In another specific embodiment, thesenolytic agent reduces the amount of fibrotic pulmonary tissue in thelung. In another specific embodiment, the senolytic agent isadministered intranasally, by inhalation, intratracheally, or byintubation. In another specific embodiment, the senescence associateddisease or disorder is atherosclerosis, and wherein the senolytic agentincreases the stability of atherosclerotic plaque. In another specificembodiment, the senescence associated disease or disorder isatherosclerosis, and wherein the senolytic agent inhibits formation ofatherosclerotic plaque in a blood vessel of the subject. In anotherspecific embodiment, the senescence associated disease or disorder isatherosclerosis, and wherein the senolytic agent reduces the lipidcontent of an atherosclerotic plaque in a blood vessel of the subject.In another specific embodiment, the senescence associated disease ordisorder is atherosclerosis, and wherein the senolytic agent increasesthe fibrous cap thickness of the plaque. In another specific embodiment,the senescent cells are senescent preadipocytes, senescent endothelialcells, senescent fibroblasts, senescent neurons, senescent epithelialcells, senescent mesenchymal cells, senescent smooth muscle cells,senescent macrophages, or senescent chondrocytes. In another specificembodiment, the senolytic agent kills at least 20% of the senescentcells and kills no more than 5% of non-senescent cells in an organ ortissue comprising the senescent cells associated with the senescenceassociated disease or disorder. In another specific embodiment, thesenolytic agent kills at least 25% of the senescent cells in an organ ortissue comprising the senescent cells associated with the senescenceassociated disease or disorder.

In one embodiment, a method is provided for treating osteoarthritis in asubject comprising administering to the subject atherapeutically-effective amount of a small molecule senolytic agentthat selectively kills senescent cells over non-senescent cells, wherein(a) the senolytic agent is administered in at least two treatment cycleswherein each treatment cycle independently comprises a treatment courseof from 1 day to 3 months followed by a non-treatment interval, andwherein the non-treatment interval is at least two weeks; or (b) thesenolytic agent is administered directly to the osteoarthritic joint. Inanother specific embodiment, the senolytic agent induces collagen TypeII production in the osteoarthritic joint. In another specificembodiment, senolytic agent inhibits erosion of a proteoglycan layer inthe osteoarthritic joint. In another specific embodiment, the senolyticagent inhibits erosion of a bone of the osteoarthritic joint. Alsoprovided herein in an embodiment, is a method for inducing production ofcollagen Type II comprising administering to a subject in need thereof atherapeutically-effective amount of a senolytic agent, which selectivelykills senescent cells over non-senescent cells, wherein (a) thesenolytic agent is administered in at least two treatment cycles whereineach treatment cycle independently comprises a treatment course of from1 day to 3 months followed by a non-treatment interval, wherein thenon-treatment interval is at least two weeks; or (b) the senolytic agentis administered directly to the osteoarthritic joint. In anotherspecific embodiment, the senolytic agent is administeredintra-articularly In another specific embodiment, the senolytic agent isadministered topically, transdermally, or intradermally. In anotherspecific embodiment, the senolytic agent is administered as a bolusinfusion. In another specific embodiment, the senolytic agent iscombined with at least one pharmaceutical excipient to formulate apharmaceutical composition that provides timed release of the senolyticagent. In another specific embodiment, the senolytic agent inhibitserosion of a proteoglycan layer in the osteoarthritic joint. In anotherspecific embodiment, the senolytic agent inhibits erosion of a bone ofthe osteoarthritic joint. In another specific embodiment, the senolyticagent kills at least 20% of the senescent cells and kills no more than5% of non-senescent cells in the osteoarthritic joint. In anotherspecific embodiment, the senolytic agent kills at least 25% of thesenescent cells in the osteoarthritic joint.

In one embodiment, a method is provided for treating asenescence-associated pulmonary disease or disorder in a subjectcomprising administering to the subject a therapeutically effectiveamount of a small molecule senolytic agent that selectively killssenescent cells over non-senescent cells, wherein the senolytic agent isadministered as a monotherapy in at least two treatment cycles whereineach treatment cycle independently comprises a treatment course of from1 day to 3 months followed by a non-treatment interval wherein thenon-treatment interval is at least 2 weeks. In another specificembodiment, a method is provided for treating a senescence-associatedpulmonary disease or disorder in a subject comprising administering tothe subject a senolytic agent, which senolytic agent is a small moleculecompound that selectively kills senescent cells, wherein the senolyticagent is administered in at least two treatment cycles, each cyclecomprising a treatment course and a non-treatment interval, and whereinthe non-treatment interval is at least 2 months. In a specificembodiment, the senescence-associated pulmonary disease or disorder ispulmonary fibrosis. In another specific embodiment, pulmonary fibrosisis idiopathic pulmonary fibrosis. In another specific embodiment, thesenescence-associated pulmonary disease or disorder is chronicobstructive pulmonary disease (COPD). In another specific embodiment,the senescence-associated pulmonary disease or disorder is selected fromage-related loss of pulmonary function, cystic fibrosis, bronchiectasis,emphysema, and asthma. In another specific embodiment, the senolyticagent is administered directly to an affected pulmonary tissue thatcomprises the senescent cells. In another specific embodiment, thesenolytic agent is administered by inhalation, intranasally,intratracheally, or by intubation In another specific embodiment, thesenolytic agent is administered as a bolus infusion. In another specificembodiment, the senolytic agent is combined with at least onepharmaceutical excipient to formulate a pharmaceutical composition thatprovides timed release of the senolytic agent. In another specificembodiment, the senolytic agent kills at least 20% of the senescentcells and kills no more than 5% of non-senescent cells in a lung of thesubject. In another specific embodiment, the senolytic agent kills atleast 25% of the senescent cells in a lung of the subject.

In one embodiment, a method is provided for treating a cardiovasculardisease or disorder caused by or associated with arteriosclerosis in asubject comprising administering to the subject atherapeutically-effective amount of a small molecule senolytic agentthat selectively kills senescent cells over non-senescent cells, whereinthe senolytic agent is administered in at least two treatment cycleswherein each treatment cycle independently comprises a treatment coursefrom 1 day to 3 months followed by a non-treatment interval, wherein thenon-treatment interval is at least 2 weeks. In a specific embodiment,the subject has atherosclerosis, congestive heart failure, peripheralvascular disease, hypertension, or coronary artery disease. In anotherspecific embodiment, the cardiovascular disease or disorder isatherosclerosis. In another specific embodiment, the senolytic agentincreases the stability of atherosclerotic plaque. In another specificembodiment, the senolytic agent reduces the lipid content of anatherosclerotic plaque in a blood vessel of the subject. In anotherspecific embodiment, the senolytic agent increases the fibrous capthickness of the plaque. In another specific embodiment, the senolyticagent inhibits formation of atherosclerotic plaque in a blood vessel ofthe subject. In another specific embodiment, the likelihood ofoccurrence of myocardial infarction, angina, stroke, carotid thrombosis,or coronary thrombosis is reduced. In another embodiment, a method isprovided for increasing the stability of atherosclerotic plaque presentin a blood vessel of a subject comprising administering to the subject atherapeutically-effective amount of a small molecule senolytic agentthat selectively kills senescent cells over non-senescent cells, whereinthe senolytic agent is administered in at least two treatment cycleswherein each treatment cycle independently comprises a treatment courseof from 1 day to 3 months followed by a non-treatment interval, whereinthe non-treatment interval is at least 2 weeks. In a specificembodiment, the subject has a cardiovascular disease selected fromatherosclerosis, congestive heart failure, peripheral vascular disease,hypertension, or coronary artery disease. In another specificembodiment, the cardiovascular disease or disorder is atherosclerosis.In another specific embodiment, the senolytic agent reduces the lipidcontent of an atherosclerotic plaque in a blood vessel of the subject.In another specific embodiment, the senolytic agent increases thefibrous cap thickness of the plaque. In another specific embodiment, thesenolytic agent inhibits formation of atherosclerotic plaque in a bloodvessel of the subject. In another specific embodiment, the senolyticagent reduces the amount of atherosclerotic plaque in a blood vessel ofthe subject. In another specific embodiment, the senolytic agent isadministered parenterally or orally. In another specific embodiment, thesenolytic agent is administered directly to an artery that comprises thesenescent cells. In another specific embodiment, the senolytic agent isadministered as a bolus infusion. In another specific embodiment, thesenolytic agent is combined with at least one pharmaceutical excipientto formulate a pharmaceutical composition that provides timed release ofthe senolytic agent. In another specific embodiment, the senolytic agentkills at least 20% of the senescent cells and kills no more than 5% ofnon-senescent cells in an arteriosclerotic artery of the subject. Inanother specific embodiment, the senolytic agent kills at least 25% ofthe senescent cells in an arteriosclerotic artery of the subject.

In certain embodiments of the methods described herein and above, thetreatment course is no longer than one month or no longer than twomonths. In another specific embodiment, the treatment course is (a) nolonger than 5 days, (b) no longer than 7 days, (c) no longer than 10days, (d) no longer than 14 days, or (e) no longer than 21 days. Inanother specific embodiment, the senolytic agent is administered every2^(nd) day or every 3^(rd) day of the treatment course. In anotherspecific embodiment, the treatment course is one day, two days, threedays, or four days. In another specific embodiment, the senolytic agentis administered daily during the treatment course. In another specificembodiment, the non-treatment interval is (a) at least one month, (b) atleast 2 months, (c) at least 3 months, (d) at least 6 months, (e) atleast 9 months, or (f) at least 1 year. In another specific embodiment,the treatment course is one day and the non-treatment interval isbetween 0.5-12 months. In other particular embodiments, when an MDM2inhibitor is administered, the treatment course is at least 5 days. Inanother specific embodiment, the senolytic agent is administered as amonotherapy. In another specific embodiment, the senolytic agent isadministered in three or more treatment cycles.

In certain embodiments related to the methods described above and hereinthe senolytic agent is selected from an MDM2 inhibitor; an inhibitor ofone or more BCL-2 anti-apoptotic protein family members wherein theinhibitor inhibits at least BCL-xL; and an Akt specific inhibitor. Inanother specific embodiment, the MDM2 inhibitor is a cis-imidazolinecompound, a spiro-oxindole compound, or a benzodiazepine compound. Inanother specific embodiment, the cis-imidazoline compound is a nutlincompound. In another specific embodiment, the nutlin compound isNutlin-3a. In another specific embodiment, the cis-imidazoline compoundis RG-7112, RG7388, RO5503781, or is a dihydroimidazothiazole compound.In another specific embodiment, the dihydroimidazothiazole compound isDS-3032b. In another specific embodiment, the MDM2 inhibitor is aspiro-oxindole compound selected from MI-63, MI-126, MI-122, MI-142,MI-147, MI-18, MI-219, MI-220, MI-221, MI-773, and3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one.In another specific embodiment, the MDM2 inhibitor is Serdemetan; apiperidinone compound; CGM097; or an MDM2 inhibitor that also inhibitsMDMX and which is selected from RO-2443 and RO-5963. In another specificembodiment, the piperidinone compound is AM-8553. In another specificembodiment, the inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members is a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-winhibitor; or a BCL-xL selective inhibitor. In another specificembodiment, the BCL-xL selective inhibitor is a benzothiazole-hydrazonecompound, an aminopyridine compound, a benzimidazole compound, atetrahydroquinolin compound, or a phenoxyl compound. In another specificembodiment, the benzothiazole-hydrazone compound is a WEHI-539. Inanother specific embodiment, the inhibitor of the one or more BCL-2anti-apoptotic protein family members is A-1155463, ABT-263, or ABT-737.In another specific embodiment, the Akt inhibitor is MK-2206 In anotherspecific embodiment, the senolytic agent is an MDM2 inhibitor or is aninhibitor of one or more BCL-2 anti-apoptotic protein family memberswherein the inhibitor inhibits at least BCL-xL and is cytotoxic tocancer cells, the total dose of the senolytic agent administered duringeach treatment cycle is an amount ineffective for treating a cancer. Inanother specific embodiment, the MDM2 inhibitor is Nutlin-3a; RG-7112;RG7388; RO5503781; DS-3032b; MI-63; MI-126; MI-122; MI-142; MI-147;MI-18; MI-219; MI-220; MI-221; MI-773; and3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one;Serdemetan; AM-8553; CGM097; or an MDM2 inhibitor that also inhibitsMDMX and which is selected from RO-2443 and RO-5963. In another specificembodiment, the inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members is ABT-263, ABT-737, A-1155463, or WEHI-539.

Also provided herein in another embodiment, is a method for treating asenescence-associated disease or disorder in a subject comprisingadministering to the subject a senolytic agent that is a small moleculeMDM2 inhibitor that selectively kills senescent cells over non-senescentcells, wherein the senolytic agent is administered as a monotherapy,wherein the senolytic agent is administered in at least two treatmentcycles wherein each treatment cycle independently comprises a treatmentcourse followed by a non-treatment interval, wherein the treatmentcourse is at least 5 days long and no longer than three months, duringwhich treatment course the MDM2 inhibitor is administered on at least 5days, and wherein the senescence-associated disease or disorder is not acancer. In a specific embodiment, the treatment course is at least 9days long. In another specific embodiment, the treatment course is nolonger than one month or no longer than two months. In another specificembodiment, the treatment course is no longer than 10, 14, or 21 days.In another specific embodiment, the MDM2 inhibitor is administereddaily. In another specific embodiment, the MDM2 inhibitor isadministered every 2^(nd) day or every 3^(rd) day of the treatmentcourse. In another specific embodiment, the non-treatment interval is atleast 2 weeks, at least one month, at least 2 months, at least 6 months,at least 9 months, or at least 1 year. In another specific embodiment,the MDM2 inhibitor to the subject comprises three or more treatmentcycles. In another specific embodiment, the MDM2 inhibitor is acis-imidazoline compound, a spiro-oxindole compound, or a benzodiazepinecompound. In another specific embodiment, the cis-imidazoline compoundis a nutlin compound. In another specific embodiment, the nutlincompound is Nutlin-3a. In another specific embodiment, thecis-imidazoline compound is RG-7112, RG7388, or RO5503781, or adihydroimidazothiazole compound. In another specific embodiment, thedihydroimidazothiazole compound is DS-3032b. In another specificembodiment, the MDM2 inhibitor is a spiro-oxindole compound selectedfrom MI-63, MI-126; MI-122, MI-142, MI-147, MI-18, MI-219, MI-220,MI-221, MI-773, and3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one.In another specific embodiment, the MDM2 inhibitor is Serdemetan; apiperidinone compound; an MDM2 inhibitor that also inhibits MDMX and isselected from RO-2443 and RO-5963; or CGM097. In another specificembodiment, the piperidinone compound is AM-8553. In another specificembodiment, the method further comprises administering to the subject asmall molecule inhibitor of one or more of mTOR, NFκB, PI3-k, and AKTpathways. In another specific embodiment, the method further comprisesadministering to the subject an Akt specific inhibitor. In anotherspecific embodiment, the method further comprises the AKT inhibitor isMK-2206.

In one embodiment, a method is provided for treating asenescence-associated disease or disorder in a subject comprisingadministering to the subject a senolytic agent that is a small moleculeinhibitor of one or more BCL-2 anti-apoptotic protein family memberswherein the inhibitor inhibits at least BCL-XL, wherein the senolyticagent selectively kills senescent cells over non-senescent cells,wherein the senolytic agent is administered in at least two treatmentcycles, wherein each treatment cycle independently comprises a treatmentcourse of from 1 day to 3 months followed by a non-treatment interval ofat least 2 weeks, and wherein the senescence-associated disease ordisorder is not a cancer. In another specific embodiment, the inhibitorof one or more BCL-2 anti-apoptotic protein family members is aBCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-w inhibitor; or a BCL-xLselective inhibitor. In another specific embodiment, the inhibitor ofthe one or more BCL-2 anti-apoptotic protein family members is abenzothiazole-hydrazone compound, an aminopyridine compound, abenzimidazole compound, a tetrahydroquinolin compound, or a phenoxylcompound. In another specific embodiment, the benzothiazole-hydrazonecompound is a WEHI-539. In another specific embodiment, the inhibitor ofthe one or more BCL-2 anti-apoptotic protein family members isA-1155463, ABT-263, or ABT-737. In another specific embodiment, themethod further comprises administering to the subject a small moleculeinhibitor of one or more of mTOR, NFκB, PI3-k, and AKT pathways. Inanother specific embodiment, the method further comprises administeringto the subject an Akt specific inhibitor. In another specificembodiment, the method further comprises the AKT inhibitor is MK-2206.

In one embodiment, a method is provided for treating asenescence-associated disease or disorder in a subject comprisingadministering to the subject a senolytic agent that is a small moleculespecific inhibitor of AKT, wherein the senolytic agent wherein thesenolytic agent selectively kills senescent cells over non-senescentcells, wherein the senolytic agent is administered as a monotherapy inat least two treatment cycles, wherein each treatment cycleindependently comprises a treatment course of from 1 day to 3 monthsfollowed by a non-treatment interval of at least 2 weeks, and whereinthe senescence-associated disease or disorder is not a cancer. Inanother specific embodiment, the AKT inhibitor is MK-2206. In anotherspecific embodiment, the method further comprises administering to thesubject a small molecule inhibitor of one or more of mTOR, NFκB, andPI3-k, pathways.

In other specific embodiments of the methods described above and herein,each treatment course is no longer than one month or no longer than twomonths. In another specific embodiment, each treatment course is (a) nolonger than 5 days, is (b) no longer than 7 days, is (c) no longer than10 days, is (d) no longer than 14 days, or is (e) no longer than 21days. In another specific embodiment, the senolytic agent isadministered every 2^(nd) day or every 3^(rd) day of the treatmentcourse. In another specific embodiment, each treatment course is oneday, two days, three days, or four days. In another specific embodiment,the senolytic agent is administered daily during the treatment course.In another specific embodiment, the non-treatment interval is at leasttwo weeks, one month, at least 2 months, at least 6 months, at least 9months, or at least 1 year. In another specific embodiment, thesenolytic agent to the subject comprises three or more treatment cycles.In another specific embodiment, the senolytic agent is administered as amonotherapy. In another specific embodiment, the senescence-associateddisease or disorder is a cardiovascular disease selected fromatherosclerosis, angina, arrhythmia, cardiomyopathy, congestive heartfailure, coronary artery disease, carotid artery disease, endocarditis,coronary thrombosis, myocardial infarction, hypertension, aorticaneurysm, cardiac diastolic dysfunction, hypercholesterolemia,hyperlipidemia, mitral valve prolapsed, peripheral vascular disease,cardiac stress resistance, cardiac fibrosis, brain aneurysm, and stroke.In another specific embodiment, the subject has a cardiovascular diseaseselected from atherosclerosis, congestive heart failure, peripheralvascular disease, hypertension, or coronary artery disease. In anotherspecific embodiment, the senescence-associated disease or disorder isinflammatory or autoimmune disease or disorder selected fromosteoarthritis, osteoporosis, oral mucositis, inflammatory boweldisease, kyphosis, and herniated intervertebral disc In another specificembodiment, the senescence-associated disease or disorder is aneurodegenerative disease selected from Alzheimer's disease, Parkinson'sdisease, Huntington's disease, dementia, mild cognitive impairment, andmotor neuron dysfunction. In another specific embodiment, thesenescence-associated disease or disorder is a metabolic diseaseselected from diabetes, diabetic ulcer, metabolic syndrome, and obesity.In another specific embodiment, the senescence-associated disease ordisorder is a pulmonary disease selected from idiopathic pulmonaryfibrosis, chronic obstructive pulmonary disease, asthma, cysticfibrosis, emphysema, bronchiectasis, and age-related loss of pulmonaryfunction. In another specific embodiment, the senescence-associateddisease or disorder is an eye disease or disorder selected from maculardegeneration, glaucoma, cataracts, and vision loss. In another specificembodiment, the senescence-associated disease or disorder is anage-related disorder selected from renal disease, renal failure,frailty, hearing loss, muscle fatigue, skin conditions, skin woundhealing, liver fibrosis, pancreatic fibrosis, oral submucosa fibrosis,and sarcopenia. In another specific embodiment, thesenescence-associated disease or disorder is a dermatological disease ordisorder is selected from eczema, psoriasis, hyperpigmentation, nevi,rashes, atopic dermatitis, urticaria, diseases and disorders related tophotosensitivity or photoaging, rhytides; pruritis; dysesthesia;eczematous eruptions; eosinophilic dermatosis; reactive neutrophilicdermatosis; pemphigus; pemphigoid; immunobullous dermatosis;fibrohistocytic proliferations of skin; cutaneous lymphomas; andcutaneous lupus. In another specific embodiment, thesenescence-associated disease or disorder is atherosclerosis;osteoarthritis; idiopathic pulmonary fibrosis; or chronic obstructivepulmonary disease. In another specific embodiment, n thesenescence-associated disease or disorder is osteoarthritis and thesenolytic agent is administered directly to the osteoarthritic joint. Inanother specific embodiment, the senolytic agent is administeredintra-articularly to the osteroarthritic joint. In another specificembodiment, the senolytic agent is administered topically,transdermally, or intradermally. In another specific embodiment, thesenescence-associated disease or disorder is osteoarthritis and thesenolytic agent induces production of Type II collagen in a joint. Inanother specific embodiment, the senescence-associated disease ordisorder is osteoarthritis and the senolytic agent inhibits erosion of aproteoglycan layer in a joint. In another specific embodiment, thesenescence-associated disease or disorder is osteoarthritis and thesenolytic agent inhibits erosion of a bone of a joint. In anotherspecific embodiment, the senescence-associated disease or disorder isidiopathic pulmonary fibrosis and the senolytic agent reduces the amountof fibrotic pulmonary tissue in the lung. In another specificembodiment, the senolytic agent is administered intranasally, byinhalation, intratracheally, or by intubation. In another specificembodiment, the senolytic agent is combined with at least onepharmaceutically acceptable excipient to formulate a pharmaceuticallyacceptable composition to provide timed-release of the senolytic agent.In another specific embodiment, the senolytic agent is administered as abolus infusion. In another specific embodiment, thesenescence-associated disease or disorder is atherosclerosis, andwherein the senolytic agent increases stability of atheroscleroticplaque. In another specific embodiment, the senescence-associateddisease or disorder is atherosclerosis, and wherein the senolytic agentinhibits formation of atherosclerotic plaque in a blood vessel of thesubject. In another specific embodiment, the senescence-associateddisease or disorder is atherosclerosis, and wherein the senolytic agentreduces the lipid content of an atherosclerotic plaque in a blood vesselof the subject. In another specific embodiment, thesenescence-associated disease or disorder is atherosclerosis, andwherein the senolytic agent increases the fibrous cap thickness of theplaque. In another specific embodiment, the senescence-associateddisease or disorder is atherosclerosis, and wherein the likelihood ofoccurrence of myocardial infarction, angina, stroke, carotid thrombosis,or coronary thrombosis is reduced. In another specific embodiment, thesenescent cells are senescent preadipocytes, senescent endothelialcells, senescent fibroblasts, senescent neurons, senescent epithelialcells, senescent mesenchymal cells, senescent smooth muscle cells,senescent macrophages, or senescent chondrocytes. In another specificembodiment, the senolytic agent kills at least 20% of the senescentcells and kills no more than 5% of non-senescent cells. In anotherspecific embodiment, the senolytic agent kills at least 25% of thesenescent cells.

In one embodiment, a method is provided herein for inhibiting metastasisin a subject who has a cancer, comprising administering to the subject asingle small molecule senolytic agent that selectively kills senescentcells over non-senescent cells, wherein the senolytic agent isadministered to the subject on one or more days beginning on at leastthe sixth day subsequent to an administration cycle of a chemotherapyand not concurrent with the chemotherapy, and wherein the senolyticagent is not a chemotherapeutic agent for treating the cancer andwherein the senolytic agent is selected from an MDM2 inhibitor; aninhibitor of one or more BCL-2 anti-apoptotic protein family memberswherein the inhibitor inhibits at least BCL-XL selected from aBCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-w inhibitor; and a BCL-xLselective inhibitor; and an Akt specific inhibitor. In a specificembodiment, metastasis is metastasis of melanoma cells, prostate cancercells, testicular cancer cells, breast cancer cells, brain cancer cells,pancreatic cancer cells, colon cancer cells, thyroid cancer cells,stomach cancer cells, lung cancer cells, ovarian cancer cells, Kaposi'ssarcoma cells, skin cancer cells, renal cancer cells, head or neckcancer cells, throat cancer cells, squamous carcinoma cells, bladdercancer cells, osteosarcoma cells, cervical cancer cells, endometrialcancer cells, esophageal cancer cells, liver cancer cells, or kidneycancer cells. In another specific embodiment, the MDM2 inhibitor to thesubject comprises three or more treatment cycles. In another specificembodiment, the MDM2 inhibitor is a cis-imidazoline compound, aspiro-oxindole compound, or a benzodiazepine compound. In anotherspecific embodiment, the cis-imidazoline compound is a nutlin compound.In another specific embodiment, the nutlin compound is Nutlin-3a. Inanother specific embodiment, the cis-imidazoline compound is RG-7112,RG7388, or RO5503781, or a dihydroimidazothiazole compound. In anotherspecific embodiment, the dihydroimidazothiazole compound is DS-3032b. Inanother specific embodiment, the MDM2 inhibitor is a spiro-oxindolecompound selected from MI-63, MI-126; MI-122, MI-142, MI-147, MI-18,MI-219, MI-220, MI-221, MI-773, and3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one.In another specific embodiment, the MDM2 inhibitor is Serdemetan; apiperidinone compound; an MDM2 inhibitor that also inhibits MDMX and isselected from RO-2443 and RO-5963; or CGM097. In another specificembodiment, the piperidinone compound is AM-8553. In another specificembodiment, the inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members is a BCL-2/BCL-xL inhibitor; a BCL-2/BCL-xL/BCL-winhibitor; or a BCL-xL selective inhibitor. In another specificembodiment, the inhibitor of the one or more BCL-2 anti-apoptoticprotein family members is a benzothiazole-hydrazone compound, anaminopyridine compound, a benzimidazole compound, a tetrahydroquinolincompound, or a phenoxyl compound. In another specific embodiment, thebenzothiazole-hydrazone compound is a WEHI-539. In another specificembodiment, the inhibitor of the one or more BCL-2 anti-apoptoticprotein family members is A-1155463, ABT-263, or ABT-737. In anotherspecific embodiment, senolytic agent is an AKT inhibitor. In stillanother particular embodiment, the AKT inhibitor is MK-2206.

In another embodiment, a method is provided for identifying a senolyticagent comprising (a) inducing cells to senesce to provide establishedsenescent cells; (b) contacting a sample of the senescent cells with acandidate agent and contacting a sample of control non-senescent cellswith the candidate agent; (c) determining the level of survival of thesenescent cells and the level of survival of the non-senescent cellswherein when the level of survival of the senescent cells is less thanthe level of survival of the non-senescent cells, the candidate agent isa senolytic agent. In a specific embodiment, the method furthercomprises contacting the senolytic agent identified in step (c) andcells capable of producing collagen; and determining the level ofcollagen produced by the cells, thereby identifying a senolytic agentfor treating osteoarthritis. In a specific embodiment, the cells capableof producing collagen are chondrocytes. In a specific embodiment, thecollagen produced is Type 2 collagen. In a specific embodiment, themethod further comprises administering the senolytic agent to anon-human animal with osteoarthritic lesions in a joint and determiningone or more of (a) the level of senescent cells in the joint; (b)physical function of the animal; (c) the level of one or more markers ofinflammation; (d) histology of the joint; and (e) the level of Type 2collagen produced, thereby determining therapeutic efficacy of thesenolytic agent wherein one or more of the following is observed in thetreated animal compared with an animal not treated with the senolyticagent: (i) a decrease in the level of senescent cells in the joint ofthe treated animal; (ii) improved physical function of the treatedanimal; (iii) a decrease in the level of one or more markers ofinflammation in the treated animal; (iv) increased histological normalcyin the joint of the treated animal; and (v) an increase in the level ofType 2 collagen produced in the treated animal. In a specificembodiment, the method further comprises administering the senolyticagent to a non-human animal of an atherosclerosis animal model, whichanimal has atherosclerotic plaques, and determining one or more of (a)the level of one or more markers of inflammation; and (b) the level ofatherosclerotic plaque thereby determining therapeutic efficacy of thesenolytic agent wherein one or more of the following is observed in thetreated animal compared with an animal not treated with the senolyticagent: (i) a decrease in the level of one or more markers ofinflammation in the treated animal; and (ii) a decrease in the level ofatherosclerotic plaques in the treated animal; thereby identifying asenolytic agent for treating atherosclerosis. In a specific embodiment,the method further comprises administering the senolytic agent tonon-human animal of pulmonary disease animal model, which animal haspulmonary fibrotic tissue, and determining one or more of (a) the levelof one or more markers of inflammation; and (b) the level of pulmonaryfibrotic tissue thereby determining therapeutic efficacy of thesenolytic agent wherein one or more of the following is observed in thetreated animal compared with an animal not treated with the senolyticagent: (i) a decrease in the level of one or more markers ofinflammation in the treated animal; and (ii) a decrease in the level ofpulmonary fibrotic tissue in the treated animal, thereby identifying asenolytic agent for treating a senescence-associated pulmonary disease.

In another embodiment, a method is provided for treating asenescence-associated disease or disorder in a subject comprising: (a)detecting the level of senescent cells in the subject; and (b)administering to the subject a senolytic agent that selectively killssenescent cells, wherein the senolytic agent is selected from a smallmolecule and is selected from an MDM2 inhibitor, an Akt specificinhibitor, an inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members wherein the inhibitor inhibits at least BCL-xL. In aspecific embodiment, the method further comprises the inhibitor of oneor more BCL-2 anti-apoptotic protein family members is aBcl-2/Bcl-xL/Bcl-w inhibitor, a Bcl-2/Bcl-xL inhibitor, a Bcl-xL/Bcl-winhibitor, or a Bcl-xL selective inhibitor.

In other particular embodiments, a method is provided for treating,reducing the likelihood of occurrence of, or delaying onset of asenescent cell-associated disease or disorder in a subject who has asenescent cell-associated disease or disorder or who has at least onepredisposing factor for developing the senescent cell-associated diseaseor disorder, comprising administering to the subject a senolytic agentthat alters either one or both of a cell survival signaling pathway andan inflammatory pathway in the senescent cell, thereby promoting deathof the senescent cell, with the proviso that if the subject has acancer, the senolytic agent is not a primary therapy for treating thecancer, wherein the senolytic agent is administered once every 0.5-12months, and wherein the senescent cell-associated disease or disorder isa cardiovascular disease or disorder, inflammatory disease or disorder,a pulmonary disease or disorder, a neurological disease or disorder, achemotherapeutic side effect, a radiotherapy side effect, or metastasis.In another specific embodiment, a method is provided for treating,reducing the likelihood of occurrence of, or delaying onset of asenescent cell-associated disease or disorder in a subject who has asenescent cell-associated disease or disorder or who has at least onepredisposing factor for developing the senescent cell-associated diseaseor disorder, comprising administering to the subject a senolytic agentthat alters either one or both of a cell survival signaling pathway andan inflammatory pathway in the senescent cell, thereby promoting deathof the senescent cell, wherein the senolytic agent is administered onceevery 4-12 months.

Also provided herein are uses of the senolytic agents described herein.In one embodiment, a use is provided for a senolytic agent for treatinga senescence-associated disease or disorder wherein atherapeutically-effective amount of a small molecule senolytic agentthat selectively kills senescent cells over non-senescent cells issuitable for administration in at least two treatment cycles, whereineach treatment cycle independently comprises a treatment course of from1 day to 3 months followed by a non-treatment interval of at least 2weeks; provided that if the senolytic agent is an MDM2 inhibitor, theMDM2 inhibitor is administered as a monotherapy, and each treatmentcourse is at least 5 days long during which the MDM2 inhibitor isadministered on at least 5 days; wherein the senescence-associateddisease or disorder is not a cancer. The senolytic agents describedherein may be used for the manufacture of a medicament for treating asenescence-associated disease or disorder as described herein.

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments.However, one skilled in the art will understand that the invention maybe practiced without these details. In other instances, well-knownstructures have not been shown or described in detail to avoidunnecessarily obscuring descriptions of the embodiments. Unless thecontext requires otherwise, throughout the specification and claimswhich follow, the word “comprise” and variations thereof, such as,“comprises” and “comprising,” are to be construed in an open, inclusivesense, that is, as “including, but not limited to.” In addition, theterm “comprising” (and related terms such as “comprise” or “comprises”or “having” or “including”) is not intended to exclude that in othercertain embodiments, for example, an embodiment of any composition ofmatter, composition, method, or process, or the like, described herein,may “consist of” or “consist essentially of” the described features.Headings provided herein are for convenience only and do not interpretthe scope or meaning of the claimed embodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment. Thus, the appearances of the phrases “in one embodiment” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

Also, as used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “anon-human animal” may refer to one or more non-human animals, or aplurality of such animals, and reference to “a cell” or “the cell”includes reference to one or more cells and equivalents thereof (e.g.,plurality of cells) known to those skilled in the art, and so forth.When steps of a method are described or claimed, and the steps aredescribed as occurring in a particular order, the description of a firststep occurring (or being performed) “prior to” (i.e., before) a secondstep has the same meaning if rewritten to state that the second stepoccurs (or is performed) “subsequent” to the first step. The term“about” when referring to a number or a numerical range means that thenumber or numerical range referred to is an approximation withinexperimental variability (or within statistical experimental error), andthus the number or numerical range may vary between 1% and 15% of thestated number or numerical range. For example, the use of “about X”shall encompass +/−1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14% and 15% of the value X. It should also be noted that the term“or” is generally employed in its sense including “and/or” unless thecontent clearly dictates otherwise. The term, “at least one,” forexample, when referring to at least one compound or to at least onecomposition, has the same meaning and understanding as the term, “one ormore.”

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic of general timelines and procedures fortreatment with Nutlin-3a (Nut) of (1) cells induced to senesce byirradiation (Sen(IR)); (2) cells induced to senesce by treatment withdoxorubicin (Sen(Doxo)); and (3) non-senescent cells (Non Sen).

FIGS. 2A-D show the effect of Nutlin-3a on survival of fibroblastsinduced to senesce by irradiation. FIG. 2A illustrates effect ofNutlin-3a at 0, 2.5 or 10 μM after 9 days of treatment (D9) onirradiated (IR) senescent foreskin fibroblasts (Sen(IR)HCA2). FIG. 2Bshows percent survival of irradiated BJ cells (Sen(IR)BJ) treated withNutlin 3a at the concentrations shown.

FIG. 2C shows percent survival of irradiated lung fibroblasts(Sen(IR)IMR90)), and FIG. 2D shows percent survival of irradiated mouseembryonic fibroblasts (MEFs) treated with Nutlin-3a.

FIGS. 3A-B illustrate the effect of Nutlin-3a on survival of cellsinduced to senesce by treatment with doxorubicin. HCA2 cells weretreated with Nutlin-3a for 9 days (D9), and aortic endothelial cellswere treated with Nutlin-3a for 11 days (D11), and then percent survivalwas determined. FIG. 3A shows the effect of Nutlin-3a ondoxorubicin-treated (Doxo) senescent foreskin fibroblasts (HCA2). FIG.3B illustrates the effect of Nutlin-3a on doxorubicin-treated (Doxo)senescent aortic endothelial cells (Endo Aort) (FIG. 3B).

FIGS. 4A-C show percent growth of non-senescent fibroblasts treated withNutlin-3a. Cells were treated with Nutlin-3a for 9 days and percentgrowth determined (D9). Nutlin-3a was non-toxic to non-senescentforeskin fibroblasts (Non Sen HCA2) as shown in FIG. 4A, non-toxic tonon-senescent lung fibroblasts (Non Sen IMR90) as shown in FIG. 4B, andnon-toxic to non-senescent lung mouse embryonic fibroblasts (Non SenMEFs) as shown in FIG. 4C.

FIGS. 5A-B illustrate percent growth of non-senescent aortic endothelialcells and non-senescent pre-adipocytes treated with Nutlin-3a. Cellswere treated with Nutlin-3a for 11 days and percent growth determined(D11). FIG. 5A and FIG. 5B show that Nutlin-3a is non-toxic tonon-senescent aortic endothelial (Non Sen Endo Aort) cells and tonon-senescent pre-adipocytes (Non Sen Pread), respectively.

FIG. 6 presents a schematic of a timeline for treatment and imaginganalysis of p16-3MR mice with Nutlin-3a. On day 35, the mice weresacrificed and fat and skin were collected for RNA, and lungs werecollected and flash frozen for immunomicroscopy. RNA was analyzed forexpression of SASP factors (mmp3, IL-6) and senescence markers (p21,p16, and p53). Frozen lung tissue was analyzed for DNA damage marker(γH2AX).

FIG. 7 shows a schematic of p16-3MR transgene insertion. 3MR(tri-modality reporter) is a fusion protein containing functionaldomains of a synthetic Renilla luciferase (LUC), monomeric redfluorescence protein (mRFP), and truncated herpes simplex virus (HSV)-1thymidine kinase (tTK), which allows killing by ganciclovir (GCV). The3MR cDNA was inserted in frame with p16 in exon 2, creating a fusionprotein containing the first 62 amino acids of p16, but does not includethe full-length wild-type p16 protein. Insertion of the 3MR cDNA alsointroduced a stop codon in the p19^(ARF) reading frame in exon 2.

FIG. 8 illustrates the reduction of luminescence intensity ofdoxorubicin-induced senescence in mice. Female C57/B16 p16-3MR mice weretreated with doxorubicin (DOXO). Luminescence was measured 10 days laterand used as baseline for each mouse (100% intensity). Nutlin-3a (NUT)was administered intraperitoneally daily from day 10 to day 24post-doxorubicin treatment (N=9). Luminescence was then measured at day7, 14, 21, 28, 35 post-Nutlin-3a treatments, and final values calculatedas % of the baseline values. Control animals (DOXO) were injected withequal volume of PBS (N=3).

FIGS. 9A-E illustrate the level of mRNA of endogenous mmp-3, IL-6, p21,p16, and p53 in the skin and fat from animals after treatment withdoxorubicin alone (DOXO) or doxorubicin plus Nutlin-3a (DOXO+NUT). Thevalues represent the fold induction of the particular mRNA compared withuntreated control animals. FIG. 9A: p21; FIG. 9B: p16^(INK4a) (p16);FIG. 9C: p53; FIG. 9D: mmp-3; and FIG. 9E: IL-6. Data were obtained fromdoxorubicin-treated mice (Doxo N=3), and doxorubicin+Nutlin-3a-treatedmice (Doxo+Nutlin N=6).

FIGS. 10A-B present data showing that Nutlin-3a reduced the number ofcells with doxorubicin-induced DNA damage. FIG. 10A presentsimmunofluorescence microscopy of lung sections from doxorubicin treatedanimals (DOXO) (left panel) and doxorubicin and Nutlin-3a-treated mice(DOXO+NUTLIN) detected by binding to a primary rabbit polyclonalantibody specific for γH2AX followed by incubation with a secondary goatanti-rabbit antibody, and then counterstained with DAPI. FIG. 10B showsthe percent positive cells from immunofluorescence microscopy calculatedand represented as percentage of the total number of cells. Data wereobtained from doxorubicin-treated mice (Doxo N=3), anddoxorubicin+Nutlin-3a-treated mice (Doxo-Nutlin N=3).

FIG. 11 shows that Nutlin-3a treatment reduced senescence-associated(SA) 3-galactosidase (β-gal) intensity of fat biopsies from animalsfirst treated with doxorubicin. Female C57/BL6 p16-3MR mice were treatedwith doxorubicin. A portion of the doxorubicin treated animals receivedNutlin-3a (NUT) or PBS (DOXO) daily from day 10 to day 24post-doxorubicin treatment. Three weeks after the Nutlin-3a treatment,mice were sacrificed and fat biopsies immediately fixed and stained witha solution containing X-Gal. Untreated animals were used as negativecontrol (CTRL).

FIGS. 12A-12C show detection of IL-6 production in nuclei ofnon-senescent (NS) cells and irradiated (IR) senescent cells treatedwith Nutlin-3a. Primary human fibroblast (IMR90) cells were irradiatedat Day −6 and treated with 10 μM Nutlin-3a or DMSO (vehicle control) inmedia from Day 0 to Day 9. Cells were cultured for an additional 6 daysin media without Nutlin-3a or DMSO (Day 12 and Day 15). IL-6 wasdetected with an anti-IL-6 antibody in nuclei of cells at Day 9 and atDay 12. The percent IL-6 positive nuclei in each of irradiated Nutlin-3atreated cells and DMSO treated cells is illustrated in FIG. 12A.Immunofluorescence of cells expressing IL-6 detected with an anti-IL-6antibody is illustrated in FIG. 12B. FIG. 12C illustrates the relativelevel of IL-6 secretion in senescent cells treated with Nutlin-3a (Sen(IR) Nut3a 10 μM) or vehicle (Sen (IR) DMSO) at Days 9, 12 and 15 (D9,D12, D15, respectively). The fold increase compared to non-senescentcells (Fold NS, y-axis) is shown.

FIGS. 13A-13F illustrate the level of senescence associated proteins(p21, p16, and IL-1a) and SASP factors (CXCL-1, IL-6, and IL-8)expressed by non-senescent (NS) cells and irradiated senescent cellstreated with Nutlin-3a. IMR90 cells were irradiated at Day −6 andtreated with 10 μM Nutlin-3a or DMSO (vehicle control) in media from Day0 to Day 9. Cells were cultured for an additional 6 days (Day 12 and Day15) in media without Nutlin-3a or DMSO, changing media at Day 12.Quantitative PCR was performed, and the levels of p21 (FIG. 13A,p21/actin y-axis on log scale); p16 (FIG. 13B); IL-1a (FIG. 13C); CXCL-1(FIG. 13D); IL-6 (FIG. 13E); and IL-8 (FIG. 13F) expression weredetected in non-senescent cells (NS (i.e., Day −7)) and at Day 9 (d9)and Day 12 (d12) in senescent cells treated with Nutlin-3a (Sen (IR)Nut3A) or vehicle (Sen (IR) DMSO). The data are presented relative toexpression of actin.

FIG. 14 presents an immunoblot detecting production of proteins insenescent cells treated with Nutlin-3a. IMR90 cells were irradiated atDay −6 and treated with Nutlin-3a or DMSO (vehicle control) in mediafrom Day 0 to Day 9. Cells were cultured for an additional 6 days (Day12 and Day 15) in media without Nutlin-3a or DMSO, changing media at Day12. The levels of each protein were detected using commerciallyavailable antibodies. The data are shown for non-senescent cells (NS)and for senescent cells at days 9, 12, and 15 (Xd9, Xd12, and Xd15,respectively) cultured in 10 μM Nutlin-3a (+) or vehicle (−).

FIG. 15 depicts an exemplary timeline and treatment protocol insenescent (irradiated cells) and non-senescent cells (non-radiatedcells) for a cell counting assay.

FIG. 16 depicts a graph showing the effect of ABT-263 (“Navi”) treatmenton non-senescent IMR90 cells (Non Sen IMR90).

FIG. 17 depicts a graph showing the effect of ABT-263 treatment onsenescent IMR90 cells (Sen(IR) IMR90).

FIG. 18 depicts an exemplary timeline and treatment protocol insenescent (irradiated cells) and non-senescent cells (non-radiatedcells) in a cell viability assay (CellTiter-Glo® (CTG)).

FIG. 19 illustrates a graph showing the effect of ABT-263 treatment onnon-senescent and senescent IMR90 cells.

FIG. 20 illustrates a graph showing the effect of ABT-263 treatment innon-senescent and senescent renal epithelial cells.

FIG. 21 illustrates a graph showing the effect of ABT-263 treatment innon-senescent and senescent foreskin fibroblasts (HCA2) cells.

FIG. 22 illustrates a graph showing the effect of ABT-263 treatment innon-senescent and senescent lung fibroblast cells (IMR90).

FIG. 23 illustrates a graph showing the effect of ABT-263 treatment innon-senescent and senescent pre-adipose cells.

FIG. 24 illustrates a graph showing the effect of ABT-263 treatment innon-senescent and senescent mouse embryonic fibroblasts (MEF) cells.

FIG. 25 illustrates a graph showing the effect of ABT-263 treatment innon-senescent and senescent smooth muscle cells (Smth Mscl).

FIG. 26 illustrates a graph showing the effect of ABT-199 treatment innon-senescent and senescent IMR90 cells.

FIG. 27 illustrates a graph showing the effect of ABT-199 treatment innon-senescent and senescent IMR90 cells.

FIG. 28 illustrates a graph showing the effect of Obatoclax treatment innon-senescent and senescent IMR90 cells.

FIG. 29A and FIG. 29B: FIG. 29A presents a graph showing the effect ofABT-263 (Navi) treatment in combination with 10 nM MK-2206 innon-senescent and senescent IMR90 cells. FIG. 29B illustrates percentsurvival of non-senescent IMR90 cells (IMR90 NS) and senescent IMR90cells (IMR90 Sen(IR)) when exposed to MK-2206 alone.

FIGS. 30A-B illustrate the effect of WEHI-539 on percent survival ofsenescent irradiated lung fibroblasts (Sen(IR)IMR90)) (FIG. 30A) andpercent survival of irradiated renal cells (Sen(IR)) (FIG. 30B).NS=Non-senescent cells, which were not exposed to radiation.

FIG. 31 illustrates that in the presence of a caspase inhibitor(panCaspase inhibitor, Q-VD-OPh) the senolytic activity of WEHI-539 isinhibited. The left side of FIG. 31 illustrates the effect of WEHI-539on killing senescent cells (IMR90 Sen(IR)). The data points within theboxed area depict killing of senescent cells at the WEHI-539concentrations of 1.67 μM and 5 μM of to which non-senescent cells (NS)and senescent cells (Sen (IR)) were exposed in the presence or absenceof Q-VC-OPh. The percent survival of non-senescent cells and senescentcells in the presence and absence of the pan-Caspase inhibitor (Q-VD inthe figure) is illustrated in the figure on the bottom right.

FIG. 32 shows the effect of specific shRNA molecules on survival ofsenescent cells. Senescent cells and non-senescent IMR90 cells weretransduced with lentiviral vectors comprising shRNA molecules specificfor each of BCL-2, BCL-xL, and BCL-w encoding polynucleotides. The ratioof senescent cell survival to non-senescent cell survival for each shRNAis shown. Each bar represents the average of triplicates. The shRNAsequences introduced into the cells are as follows from left to right:BCL-2: SEQ ID NO:1, 3, 3, 5; BCL-XL: SEQ ID NO: 7, 9, 11, 13; BCL-w: SEQID NO:15, 17, 19, 21; two non-transduced (NT) samples.

FIG. 33 illustrates the effect of ABT-737 on viability of non-senescentlung fibroblast cells (IMR90) (IMR90 NS) and senescent lung fibroblastcells (IMR90) (IMR90 Sen(IR)).

FIG. 34 illustrates that in the presence of a caspase inhibitor(panCaspase inhibitor, Q-VD-OPh) the senolytic activity of ABT-263 isinhibited. The top left side of FIG. 34 illustrates the effect ofABT-263 on killing senescent cells (IMR90 Sen(IR)). Non-senescent cells(NS) and senescent cells (Sen (IR)) were exposed to ABT-263 atconcentrations of 0.33 μM and 1 μM in the presence or absence of thepan-Caspase inhibitor, Q-VC-OPh. The percent survival of non-senescentcells and senescent cells in the presence and absence of the pan-Caspaseinhibitor (Q-VD in the figure) is illustrated in the FIG. 34 on thebottom right.

FIG. 35 depicts animal study designs for assessing the efficacy ofremoval of senescent cells by Nutlin-3A treatment in C57BL6/J mice or byGCV treatment in 3MR mice in inhibiting signs and progression ofosteoarthritis. Group 1 animals (16×C57BL6/J mice; 1×3MR mouse)represent the anterior cruciate ligament (ACL) control group thatundergo surgery to cut the ACL (ACL surgery or osteoarthritis surgery(OA)) of one hind limb to induce osteoarthritis. Group 1 animals receiveintra-articular injections of vehicle (10 μl) qd for 5 days during week2 post-surgery and an optional second treatment cycle at week 4post-surgery, parallel to the GCV treatment in the test animals. Group 2animals (3×3MR mice) represent one treatment group that receives ACLsurgery and intra-articular injections of GCV (2.5 μg/joint) qd for 5days during week 2 post-surgery and an optional second treatment cycleat week 4 post-surgery. Group 3 animals (12×C57BL6/J) represent a secondtreatment group that received ACL surgery and intra-articular injectionsof Nutlin-3A (5.8 μg/joint) qod for 2 weeks starting at week 3post-surgery. Group 4 animals represent a second control group having asham surgery that does not sever the ACL and receiving intra-articularinjections of vehicle (10 μl) qd for 5 days during week 2 post-surgeryand an optional second treatment cycle at week 4 post-surgery, parallelto the GCV treated 3MR mice. This study design can be adapted, such asthe dosing amount and dosing schedule (e.g., number of days), for othersenolytic agents.

FIG. 36 depicts a timeline for the animal study designs described inFIG. 35.

FIGS. 37A-C illustrate the level of senescence associated proteins (p16)and SASP factors (IL-6 and MMP13) expressed by cells from joints of micethat had osteoarthritis surgery (OA surgery), joints of mice that had OAsurgery and received Nutlin-3A treatment (Nutlin-3A), joints thatreceived sham surgery, and joints of control mice that did not receiveany surgery (control). Quantitative PCR was performed, and the levels ofp16 (FIG. 37A); IL-6 (FIG. 37B); and MMP13 (FIG. 37C) expression weredetected in cells extracted from the joints of mice with OA surgery,mice with OA surgery and Nutlin-3A treatment, sham surgery, and control(no surgery). The data are presented relative to expression of actin.The data shows that Nutlin-3A treatment clears senescent cells from thejoint.

FIG. 38 illustrates the level of type 2 collagen expressed by cells fromjoints of mice that had osteoarthritis surgery (OA surgery), joints ofmice that had OA surgery and received Nutlin-3A treatment (Nutlin-3A),joints that received sham surgery, and joints of control mice that didnot receive any surgery. Quantitative PCR was performed, and the levelsof type 2 collagen was detected in cells extracted from the joints ofmice with OA surgery, mice with OA surgery and Nutlin-3A treatment, shamsurgery, and control (no surgery). The data are presented relative toexpression of actin. The data shows that Nutlin-3A treatment drives abinitio collagen production in OA joints.

FIG. 39 illustrates incapacitance measurements 4 weeks afterosteoarthritis surgery as measured by a weight bearing test to detectwhich leg mice favored. The mice were placed in a chamber, standing with1 hind paw on each scale. The weight that was placed on each hind limbwas then measured over a 3-second period. At least 3 separatemeasurements were made for each animal at each time point, and theresult was expressed as the percentage of the weight placed on theoperated limb/the contralateral unoperated limb.

FIG. 40 depicts the results of the weight bearing test shown in FIG. 39.Osteoarthritis causes mice to favor the unoperated leg over the operatedleg (Δ). Clearing senescent with Nutlin-3A abrogates this effect (∇).

FIG. 41 depicts the results of a hotplate analysis to provide anassessment of sensitivity and reaction to pain stimulus. Paw-lickresponse time for the operated hind limb (measured in seconds) due toattainment of pain threshold after placement onto a 55° C. platform wasmeasured 4 weeks after osteoarthritis (OA) surgery. The data shows thatNutlin-3A treatment reduces response time in OA surgery mice (▴) ascompared to untreated OA surgery mice (▪).

FIG. 42 presents histopathology results from animals not treated bysurgery (No Surgery (C57B)); animals that received osteoarthritissurgery and received vehicle (OA surgery (3MR)); and animals thatreceived OA surgery and were treated with Nulin-3a (OAsurgery+Nutlin-3a). Arrows point to intact or destroyed proteoglycanlayers in the joint.

FIGS. 43A-B illustrate schematics of two atherosclerosis animal modelstudies in LDLR^(−/−) transgenic mice fed a high fat diet (HFD). Thestudy illustrated in FIG. 43A assesses the extent to which clearance ofsenescent cells from plaques in LDLR^(−/−) mice with a senolytic agent(e.g., Nutlin-3A) reduces plaque load. The study illustrated in FIG. 43Bassesses the extent to which ganciclovir-based clearance of senescentcells from LDLR^(−/−) 3MR double transgenic mice improves pre-existingatherogenic disease.

FIGS. 44A-D depict graphs of the plasma lipid levels in LDLR^(−/−) micefed a HFD after one treatment cycle of Nutlin-3A or vehicle. FIG. 44Ashows total cholesterol levels in vehicle or Nutlin-3A treatedLDLR^(−/−) mice compared to LDLR^(−/−) fed a non-HFD. FIG. 44B shows HDLlevels in vehicle or Nutlin-3A treated LDLR^(−/−) mice compared toLDLR^(−/−) fed a non-HFD. FIG. 44C shows triglyceride levels in vehicleor Nutlin-3A treated LDLR^(−/−) mice compared to LDLR^(−/−) fed anon-HFD. FIG. 44D shows vLDL/LDL/IDL levels in vehicle or Nutlin-3Atreated LDLR^(−/−) mice compared to LDLR^(−/−) fed a non-HFD.

FIGS. 45A-D illustrate RT-PCR analysis of SASP factors and senescencemarkers in aortic arches of LDLR^(−/−) mice fed a HFD after onetreatment cycle of Nutlin-3A or vehicle. FIG. 45A illustrates the aorticarch (boxed). FIG. 45B-45C show expression levels of SASP factors andsenescence markers, normalized to GAPDH and expressed as fold change vs.non-HFD, vehicle-treated, age-matched LDLR^(−/−) mice. FIG. 45D showsthe data from FIGS. 45B-C in numerical form.

FIGS. 46A-C illustrate RT-PCR analysis of SASP factors and senescencemarkers in aortic arches of LDLR^(−/−) mice fed a HFD after twotreatment cycles of Nutlin-3A or vehicle. FIGS. 46A-B expression levelsof SASP factors and senescence markers, normalized to GAPDH andexpressed as fold change vs. non-HFD, vehicle-treated, age-matchedLDLR^(−/−) mice. FIG. 46C shows the data from FIGS. 46A-B in numericalform.

FIGS. 47A-C illustrate staining analysis for aortic plaques inLDLR^(−/−) mice fed a HFD after three treatment cycles of Nutlin-3A orvehicle. FIG. 47A illustrates the aorta. FIG. 47B shows the % of theaorta covered in plaques. FIG. 47C shows Sudan IV staining of the aortato visualize the plaques and the area covered by the lipid plaque wasexpressed as a percentage of the total surface area of the aorta in eachsample.

FIGS. 48A-B depict plots of platelet (FIG. 48A) and lymphocyte counts(FIG. 48B) from LDLR^(−/−) mice fed a HFD after three treatment cyclesof Nutlin-3A or vehicle.

FIGS. 49A-49B depict plots of weight and body fat/lean tissuecomposition (%), respectively, of LDLR^(−/−) mice fed a HFD after threetreatment cycles of Nutlin-3A or vehicle.

FIG. 50 depicts a graph of the effect of clearance of senescent cellswith ganciclovir in LDLR^(−/−) and LDLR^(−/−)/3MR mice fed a HFD, asmeasured by the % of the aorta covered in plaques.

FIG. 51 depicts a graph of the effect of clearance of senescent cellswith ganciclovir in LDLR^(−/−) and LDLR^(−/−)/3MR mice fed a HFD, asmeasured by the plaque cross-sectional area of the aorta.

FIG. 52 shows the effect of senescent cell clearance on resistance tocardiac stress with aging. 12 month old INK-ATTAC transgenic mice onFVB×129Sv/E×C57BL/6 mixed of C57BL/6 pure genetic backgrounds wereinjected 3×/week with AP20187 (0.2 mg/kg for the mixed cohort and 2mg/kg for the C57BL/6 cohort, respectively). At 18 months, subsets ofmale and female mice from each cohort were subjected to a cardiac stresstest and time to cardiac arrest was recorded. Control cohort receivedinjections of vehicle.

FIG. 53 shows the RT-PCR analysis of Sur2a expression in femaleINK-ATTAC transgenic mice described in FIG. 52.

FIGS. 54A-C illustrate staining analysis for aortic plaques inLDLR^(−/−)/3MR double transgenic mice and LDLR^(−/−) control mice fed aHFD after a 100 day treatment period with ganciclovir.

FIGS. 54A-54B show Sudan IV staining of the aorta to visualize theplaques in LDLR^(−/−) control mice and LDLR^(−/−)/3MR mice,respectively. FIG. 54C shows the % of the aorta covered in plaques asmeasured by area of Sudan IV staining.

FIGS. 55A-D illustrate plaque morphology analysis in LDLR^(−/−)/3MRdouble transgenic mice and LDLR^(−/−) control mice fed a HFD after a 100day treatment period with ganciclovir. FIG. 55A and FIG. 55C show SudanIV staining of the aorta to visualize the plaques in LDLR^(−/−) controlmice and LDLR^(−/−)/3MR mice, respectively. Plaques that are circledwere harvested and cut into cross-sections and stained with tocharacterize the general architecture of the atherosclerotic plaques(FIG. 55B and FIG. 55D). “#” marks fat located on the outside of theaorta.

FIG. 56 shows that SA-β-GAL crystals localize to lipid-bearing foamcells from an atherosclerotic artery of a mouse fed a high-fat diet. Themacrophage foam cell is shown by a white dotted outline and adjacent tothe macrophage foam cell is a smooth muscle foam cell. The left boxedarea in the macrophage foam cell is magnified and shown on the upperright to illustrate lysosomes with SA-β-GAL crystals. The boxed areawithin the smooth muscle foam cell is magnified and shown on the lowerright side of the figure.

FIG. 57 presents a macrophage foam cell from an atherosclerotic arteryof a mouse fed a high-fat diet. Lipid-bearing lysosomes containingSA-β-GAL crystals are noted by the arrows. FIG. 58 shows that SA-β-GALcrystals localize in the lysozomes of smooth muscle foam cells in anatherosclerotic artery of a mouse fed a high-fat diet. The boxed area inthe lower left portion of the illustration is magnified and shown in theinsert at the top left.

FIG. 59 shows the effect of senescent cell clearance on peripheralcapillary oxygen saturation (SpO₂) in bleomycin exposed mice.

FIGS. 60A-C illustrate the effect of senescent cell clearance withganciclovir on lung function in 3MR mice exposed to bleomycin. FIG. 60Ashows the effect of ganciclovir treatment on lung elastance of 3MR miceexposed to bleomycin. FIG. 60B shows the effect of ganciclovir treatmenton dynamic lung compliance of 3MR mice exposed to bleomycin. FIG. 60Cshows the effect of ganciclovir treatment on static lung compliance of3MR mice exposed to bleomycin.

FIG. 61 shows the effect of senescent cell clearance on peripheralcapillary oxygen saturation (SpO₂) in mice after 2 months and 4 monthsof cigarette smoke (CS) exposure. AP=AP20187; GAN=ganciclovir;Navi=Navitoclax (ABT-263); and Nutlin=Nutlin 3A.

FIGS. 62A-C illustrate the effect of RG-7112 (structure shown in FIG.62A) on percent survival of senescent irradiated lung fibroblasts IMR90cells ((IMR90)Sen(IR)) and non-senescent IMR90 cells, which were notexposed to radiation (IMR90 NS) after 3 days of treatment (as shown inFIG. 62B) and after six days of treatment with RG-7112 (as shown in FIG.62C).

FIGS. 63A-B illustrates that paclitaxel induces senescence in p16-3MRmice. Groups of mice (n=4) were treated three times every two days with20 mg/kg paclitaxel or vehicle. The level of luminescence in micetreated with paclitaxel is shown in FIG. 63A. The level of mRNA in skinwas determined for each of the target genes: p16, 3MR transgene, andIL-6 in animals treated with paclitaxel as shown in FIG. 63B.

FIG. 64 shows the effect of ABT-263 on mice that were initially treatedwith paclitaxel. The schematic of the experiment performed in 3MR miceis shown at the right-hand side of the figure. Mice were first treatedwith paclitaxel, followed by treatment with either vehicle, ganciclovir(gcv) or ABT-263. Wheel counts were measured for each group of mice(n=4) treated with paclitaxel+vehicle (pacli+vehicle);paclitaxel+ganciclovir (pacli+gcv); paclitaxel+ABT-263 (pacli+ABT-263);and control animals that did not receive paclitaxel (see graph at leftside of FIG. 64).

FIG. 65 shows the level of senescence induced in groups of p16-3MRanimals (n=4) treated with chemotherapeutic drugs: thalidomide (100mg/kg; 7 daily injections); romidepsin (1 mg/kg; 3 injections);pomalidomide (5 mg/kg; 7 daily injections); lenalidomide (50 mg/kg; 7daily injections); 5-azacytidine (5 mg/kg; 3 injections) and doxorubicin(10 mg/kg; 2-4 injections during a week). The level of luminescence wasmeasured in animals treated with the drugs.

FIG. 66 shows an immunoblot showing the level of different cellularproteins in senescent and non-senescent human abdominal subcutaneouspreadipocytes. Senescence was induced as described in Example 28.Lysates were prepared at several time points after induction ofsenescence, and the level of each protein in the lysates detected at 24hours and at days 3, 5, 8, 11, 15, 20, and 25 (D3, D5, D8, D11, D15,D20, and D25).

FIG. 67 shows that groups of p16-3MR mice (n=6) fed a high fat diet(high fat) for four months have increased numbers of senescence cellscompared with mice fed a regular chow diet (chow fed) (n=6).

FIG. 68 illustrates decrease of senescent cells in adipose tissue ofp16-3MR mice fed a high fat diet for four months and then treated withganciclovir compared to mice treated with vehicle. The presence ofsenescent cells in perirenal, epididymal (Epi), or subcutaneous inguinal(Ing) adipose tissue was detected by SA-β-GAL staining.

FIGS. 69A-C show the effect of ganciclovir treatment on glucosetolerance in p16-3MR mice fed a high fat diet. A bolus of glucose wasgiven at time zero, and blood glucose was monitored for up to 2 hours todetermine efficacy of glucose disposal (FIG. 69A). This is quantified asarea under the curve (AUC), with a higher AUC indicating glucoseintolerance. The glucose tolerance test (GTT) AUC's of mice treated withganciclovir is shown in FIG. 69B. Hemoglobin A1c is shown in FIG. 69C.n=9; ANOVA.

FIGS. 70A-70B show insulin sensitivity (Insulin Tolerance Testing (ITT))of p16-3MR mice fed a high fat diet after ganciclovir administration.Blood glucose levels were measured at 0, 14, 30, 60, and 120 minutesafter the administration of glucose bolus at time zero (see FIG. 70A). Achange in insulin tolerance testing when ganciclovir was administered towild-type mice was not observed (see FIG. 70B).

FIG. 71 illustrates the effect of A-1155463 on percent survival ofsenescent irradiated lung fibroblasts (Sen(IR)IMR90)) and percentsurvival of non-senescent IMR90 cells (Sen(IR)). NS=Non-senescent cells,which were not exposed to radiation.

DETAILED DESCRIPTION

Aging is a risk factor for most chronic diseases, disabilities, anddeclining health. Senescent cells, which are cells in replicativearrest, accumulate as an individual ages and may contribute partially orsignificantly to cell and tissue deterioration that underlies aging andage related diseases. Cells may also become senescent after exposure toan environmental, chemical, or biological insult or as a result of adisease. Provided herein are methods and agents for selective killing ofsenescent cells that are associated with numerous pathologies anddiseases, including age-related pathologies and diseases. As disclosedherein, senescent cell associated diseases and disorders may be treatedor prevented (i.e., likelihood of occurrence or development is reduced)by administering at least one senolytic agent. The senescentcell-associated disease or disorder treated or prevented by the agentsand methods described herein include a cardiovascular disease ordisorder, inflammatory or autoimmune disease or disorder, a pulmonarydisease or disorder, a neurological disease or disorder, adermatological disease or disorder, a chemotherapeutic side effect, aradiotherapy side effect, or metastasis, or a metabolic disease, all ofwhich are described in greater detail herein. In certain specificembodiments, the senescent cell-associated diseases or disorders treatedor prevented by the senolytic agents and methods described hereininclude, by way of example, idiopathic pulmonary fibrosis (IPF), chronicobstructive pulmonary disease (COPD), osteoarthritis, and cardiovasculardiseases and disorders associated with arteriosclerosis, such asatherosclerosis. In certain embodiments, the senescence associateddisease or disorder is not a cancer. As described in greater detailherein, senolytic agents include, for example, MDM2 inhibitors (e.g.,nutlin 3a, RG-7112); inhibitors of one or more BCL-2 anti-apoptoticprotein family members, which inhibitors inhibit a function of at leastthe anti-apoptotic protein, BCL-xL (e.g., ABT-263, ABT-737, WEHI-539,A-1155463); and Akt specific inhibitors (e.g., MK-2206).

Senolytic agents described herein are sufficient to kill significantnumbers of senescent cells. Even though cells continue to becomesenescent in a treated subject, establishment of senescence, such asshown by the presence of a senescence-associated secretory phenotype(SASP), occurs over several days (see, e.g., Laberge et al., Aging Cell11:569-78 (2012); Coppe et al., PLoS Biol 6: 2853-68 (2008); Coppe etal. PLoS One 5:39188 (2010); Rodier et al., Nat. Cell Biol. 11:973-979;Freund et al., EMBO J. 30:1536-1548 (2011)). Use of the senolytic agentsdescribed herein, therefore, offers the advantage that these agents canbe administered less frequently, intermittently, and/or at a lower dosethan many therapeutic agents commonly used for treating these diseasesand disorders. The methods described herein describe use of such agentsas senolytic agents that may be administered less frequently,intermittently, and/or at a lower dose than when the agents are used fortreating cancer other diseases.

Senolytic Agents

A senolytic agent as used herein is an agent that “selectively”(preferentially or to a greater degree) destroys, kills, removes, orfacilitates selective destruction of senescent cells. In other words,the senolytic agent destroys or kills a senescent cell in abiologically, clinically, and/or statistically significant mannercompared with its capability to destroy or kill a non-senescent cell. Asenolytic agent is used in an amount and for a time sufficient thatselectively kills established senescent cells but is insufficient tokill (destroy, cause the death of) a non-senescent cell in a clinicallysignificant or biologically significant manner. In certain embodiments,the senolytic agents described herein alter at least one signalingpathway in a manner that induces (initiates, stimulates, triggers,activates, promotes) and results in (i.e., causes, leads to) death ofthe senescent cell. The senolytic agent may alter, for example, eitheror both of a cell survival signaling pathway (e.g., Akt pathway) or aninflammatory pathway, for example, by antagonizing a protein within thecell survival and/or inflammatory pathway in a senescent cell.

Without wishing to be bound by a particular theory, the mechanism bywhich the inhibitors and antagonists described herein selectively killsenescent cells is by inducing (activating, stimulating, removinginhibition of) an apoptotic pathway that leads to cell death.Non-senescent cells may be proliferating cells or may be quiescentcells. In certain instances, exposure of non-senescent cells to thesenolytic agent as used in the methods described herein may temporarilyreduce the capability of non-senescent cell to proliferate; however, anapoptotic pathway is not induced and the non-senescent cell is notdestroyed.

Certain senolytic agents that may be used in the methods describedherein have been described as useful for treating a cancer; however, inthe methods for treating a senescence associated disorder or disease,the senolytic agents are administered in a manner that would beconsidered different and likely ineffective for treating a cancer. Themethod used for treating a senescence associated disease or disorderwith a senolytic agent described herein may comprise one or more of adecreased daily dose, decreased cumulative dose over a single treatmentcycle, or decreased cumulative dose of the agent from multiple treatmentcycles than the dose of an agent required for cancer therapy; therefore,the likelihood is decreased that one or more adverse effects (i.e., sideeffects) will occur, which adverse effects are associated with treatinga subject according to a regimen optimized for treating a cancer. Incontrast, as a senolytic agent, the compounds described herein may beadministered at a lower dose than presently described in the art or in amanner that selectively kill senescent cells (e.g., intermittentdosing). In certain embodiments, the senolytic agents described hereinmay be administered at a lower cumulative dose per treatment course ortreatment cycle that would likely be insufficiently cytotoxic to cancercells to effectively treat the cancer. In other words, according to themethods described herein, the senolytic agent is not used in a mannerthat would be understood by a person skilled in the art as a primarytherapy for treating a cancer, whether the agent is administered aloneor together with one or more additional chemotherapeutic agents orradiotherapy to treat the cancer. Even though an agent as used in themethods described herein is not used in a manner that is sufficient tobe considered as a primary cancer therapy, the methods and senolyticcombinations described herein may be used in a manner (e.g., a shortterm course of therapy) that is useful for inhibiting metastases. A“primary therapy for cancer” as used herein means that when an agent,which may be used alone or together with one or more agents, is intendedto be or is known to be an efficacious treatment for the cancer asdetermined by a person skilled in the medical and oncology arts,administration protocols for treatment of the cancer using the agenthave been designed to achieve the relevant cancer-related endpoints. Tofurther reduce toxicity, a senolytic agent may be administered at a siteproximal to or in contact with senescent cells (not tumor cells).Localized delivery of senolytic agents is described in greater detailherein.

The senolytic agents described herein alter (i.e., interfere with,affect) one or more cellular pathways that are activated during thesenescence process of a cell. Senolytic agents may alter either a cellsurvival signaling pathway (e.g., Akt pathway) or an inflammatorypathway or alter both a cell survival signaling pathway and aninflammatory pathway in a senescent cell. Activation of certain cellularpathways during senescence decreases or inhibits the cell's capabilityto induce, and ultimately undergo apoptosis. Without wishing to be boundby theory, the mechanism by which a senolytic agent selectively killssenescent cells is by inducing (activating, stimulating, removinginhibition of) an apoptotic pathway that leads to cell death. Asenolytic agent may alter one or more signaling pathways in a senescentcell by interacting with one, two, or more target proteins in the one ormore pathways, which results in removing or reducing suppression of acell death pathway, such as an apoptotic pathway. Contacting or exposinga senescent cell to a senolytic agent to alter one, two, or morecellular pathways in the senescent cell, may restore the cell'smechanisms and pathways for initiating apoptosis. In certainembodiments, a senolytic agent is an agent that alters a signalingpathway in a senescent cell, which in turn inhibits secretion and/orexpression of one or more gene products important for survival of asenescent cell. The senolytic agent may inhibit a biological activity ofthe gene product(s) important for survival of the senescent cell.Alternatively, the decrease or reduction of the level of the geneproduct(s) in the senescent cell may alter the biological activity ofanother cellular component, which triggers, initiates, activates, orstimulates an apoptotic pathway or removes or reduces suppression of theapoptotic pathway. As described herein, the senolytic agents are thebiologically active agents and capable of selectively killing senescentcells in the absence of linkage or conjugation to a cytotoxic moiety(e.g., a toxin or cytotoxic peptide or cytotoxic nucleic acid). Thesenolytic agents are also active in selectively killing senescent cellsin the absence of linkage or conjugation to a targeting moiety (e.g., anantibody or antigen-binding fragment thereof; cell binding peptide) thatselectively binds senescent cells.

Two alternative modes of cell death can be distinguished, apoptosis andnecrosis. The term apoptosis was initially used by Kerr and colleagues(Br. J. Cancer 26:239-57 (1972)) to describe the phenomenon as a mode ofcell death morphologically distinct from coagulative necrosis. Apoptosisis typically characterized by the rounding of the cell, chromatincondensation (pyknosis), nuclear fragmentation (karyorhexis), andengulfment by neighboring cells (see, e.g., Kroemer et al., Cell DeathDiffer. 16:3-11 (2009)). Several molecular assays have been developedand are used in the art; however, the morphological changes, which aredetected by light and electron microscopy, are viewed in the art as theoptimal techniques to differentiate the two distinct modes of cell death(see, e.g., Kroemer et al., supra). Alternative cell death modes, suchas caspase-independent apoptosis-like programmed cell death (PCD),autophagy, necrosis-like PCD, and mitotic catastrophe, have also beencharacterized (see, e.g., Golstein, Biochem. Sci. 32:37-43 (2007); Leistet al., Nat. Rev. Mol. Cell Biol. 2:589-98 (2001)). See, e.g., Caruso etal., Rare Tumors 5(2): 68-71 (2013); published online 2013 Jun. 7. doi:10.3081/rt.2013.e18. Techniques and methods routinely practiced in theart and described herein (e.g., TUNEL) may be used to show thatapoptotic cell death results from contact with the senolytic agentsdescribed herein.

In certain embodiments, a senolytic agent as used in the methodsdescribed herein is a small molecule compound. These senolytic agentsthat are small molecules may also be called herein senolytic compounds.In certain embodiments, the senolytic agents that are small moleculesinclude those that are activated or that are pro-drugs which areconverted to the active form by enzymes within the cell. In a morespecific embodiment, the enzymes that convert a pro-drug to an activesenolytic form are those expressed at a higher level in senescent cellsthan in non-senescent cells.

Senolytic agents described herein that may alter at least one signalingpathway include an agent that inhibits an activity of at least one ofthe target proteins within the pathway. The senolytic agent may be aspecific inhibitor of one or more BCL-2 anti-apoptotic protein familymembers wherein the inhibitor inhibits at least BCL-xL (e.g., aBcl-2/Bcl-xL/Bcl-w inhibitor; a selective Bcl-xL inhibitor; aBcl-xL/Bcl-w inhibitor); an Akt kinase specific inhibitor; or an MDM2inhibitor. In embodiments, molecules such as quercetin (and analogsthereof), enzastaurin, and dasatinib are excluded and are not compoundsused in the methods and compositions described herein. In otherparticular embodiments, methods comprise use of at least two senolyticagents wherein at least one senolytic agent and a second senolytic agentare each different and independently alter either one or both of asurvival signaling pathway and an inflammatory pathway in a senescentcell.

Small Molecules

Senolytic agents that may be used in the methods for treating orpreventing a senescence-associated disease or disorder include smallorganic molecules. Small organic molecules (also called small moleculesor small molecule compounds herein) typically have molecular weightsless than 10⁵ daltons, less than 10⁴ daltons, or less than 10³ daltons.In certain embodiments, a small molecule senolytic agent does notviolate the following criteria more than once: (1) no more than 5hydrogen bond donors (the total number of nitrogen-hydrogen andoxygen-hydrogen bonds); (2) not more than 10 hydrogen bond acceptors(all nitrogen or oxygen atoms); (3) a molecular mass less than 500daltons; (4) an octanol-water partition coefficient[5] log P not greaterthan 5.

MDM2 Inhibitors

In certain embodiments, the senolytic agent may be an MDM2 inhibitor. AnMDM2 (murine double minute 2) inhibitor that may be used in the methodsfor selectively killing senescent cells and treating or preventing(i.e., reducing or decreasing the likelihood of occurrence ordevelopment of) a senescence-associated disease or disorder may be asmall molecule compound that belongs to any one of the following classesof compounds, for example, a cis-imidazoline compound, a spiro-oxindolecompound, a benzodiazepine compound, a piperidinone compound, atryptamine compound, and CGM097, and related analogs. In certainembodiments, the MDM2 inhibitor is also capable of binding to andinhibiting an activity of MDMX (murine double minute X, which is alsoknown as HDMX in humans). The human homolog of MDM2 is called HDM2(human double minute 2) in the art. Therefore, when a subject treated bythe methods described herein is a human subject, the compounds describedherein as MDM2 inhibitors also inhibit binding of HDM2 to one or more ofits ligands.

MDM2 is described in the art as an E3 ubiquitin ligase that can promotetumor formation by targeting tumor suppressor proteins, such as p53, forproteasomal degradation through the 26S proteasome (see, e.g., Haupt etal. Nature 387: 296-299 1997; Honda et al., FEBS Lett 420: 25-27 (1997);Kubbutat et al., Nature 387: 299-303 (1997)). MDM2 also affects p53 bydirectly binding to the N-terminal end of p53, which inhibits thetranscriptional activation function of p53 (see, e.g., Momand et al.,Cell 69: 1237-1245 (1992); Oliner et al., Nature 362: 857-860 (1993)).Mdm2 is in turn regulated by p53; p53 response elements are located inthe promoter of the Mdm2 gene (see, e.g., Barak et al., EMBO J 12:461-68(1993)); Juven et al., Oncogene 8:3411-16 (1993)); Perry et al., Proc.Natl. Acad. Sci. 90:11623-27 (1993)). The existence of this negativefeedback loop between p53 and Mdm2 has been confirmed by single-cellstudies (see, e.g., Lahav, Exp. Med. Biol. 641:28-38 (2008)). See alsoManfredi, Genes & Development 24:1580-89 (2010).

Reports have described several activities and biological functions ofMDM2. These reported activities include the following: acts as aubiquitin ligase E3 toward itself and ARRB1; permits nuclear export ofp53; promotes proteasome-dependent ubiquitin-independent degradation ofretinoblastoma RB1 protein; inhibits DAXX-mediated apoptosis by inducingits ubiquitination and degradation; component of TRIM28/KAP1-MDM2-p53complex involved in stabilizing p53; component of TRIM28/KAP1-ERBB4-MDM2complex that links growth factor and DNA damage response pathways;mediates ubiquitination and subsequent proteasome degradation of DYRK2in the nucleus; ubiquitinates IGF1R and SNAI1 and promotes them toproteasomal degradation. MDM2 has also been reported to inducemono-ubiquitination of the transcription factor FOXO4 (see, e.g.,Brenkman et al., PLOS One 3(7):e2819, doi:10.1371/journal.pone.0002819).The MDM2 inhibitors described herein may disrupt the interaction betweenMDM2 and any one or more of the aforementioned cellular components.

In one embodiment, a compound useful for the methods described herein isa cis-imidazoline small molecule inhibitor. Cis-imidazoline compoundsinclude those called nutlins in the art. Similar to other MDM2inhibitors described herein, nutlins are cis-imidazoline small moleculeinhibitors of the interaction between MDM2 and p53 (see Vassilev et al.,Science 303 (5659): 844-48 (2004)). Exemplary cis-imidazolines compoundsthat may be used in the methods for selectively killing senescent cellsand treating or preventing (i.e., reducing or decreasing the likelihoodof occurrence or development of) a senescence-associated disease ordisorder are described in U.S. Pat. Nos. 6,734,302; 6,617,346; 7,705,007and in U.S. Patent Application Publication Nos. 2005/0282803;2007/0129416; 2013/0225603. In certain embodiments, the methodsdescribed herein comprise use of a nutlin compound called Nutlin-1; or anutlin compound called Nutlin-2; or a Nutlin compound called Nutlin-3(see CAS Registry No. 675576-98-4 and No. 548472-68-0). The activeenantiomer of Nutlin-3(4-[[4S,5R)-4,5-bis(4-chlorophenyl)-4,5-dihydro-2-[4-methoxy-2-(1-methylethoxy)phenyl]-1H-imidazol-1-yl]carbonyl]-2-piperazinone)is called Nutlin-3a in the art. In certain embodiments, the methodsdescribed herein comprise use of Nutlin-3a for selectively killingsenescent cells.

Nutlin-3 is described in the art as a nongenotoxic activator of the p53pathway, and the activation of p53 is controlled by the murine doubleminute 2 (MDM2) gene. The MDM2 protein is an E3 ubiquitin ligase andcontrols p53 half-life by way of ubiquitin-dependent degradation.Nutlin-3a has been investigated in pre-clinical studies (e.g., withrespect to pediatric cancers) and clinical trials for treatment ofcertain cancers (e.g., retinoblastoma). To date in vitro andpre-clinical studies with Nutlin-3 have suggested that the compound hasvariable biological effects on the function of cells exposed to thecompound. For example, Nutlin-3 reportedly increases the degree ofapoptosis of cancer cells in hematological malignancies including B-cellmalignancies (see, e.g., Zauli et al., Clin. Cancer Res. 17:762-70(2011; online publication on Nov. 24, 2010) and references citedtherein) and in combination with other chemotherapeutic drugs, such asdasatinib, the cytotoxic effect appears synergistic (see, e.g., Zauli etal., supra).

Another exemplary cis-imidazoline small molecule compound useful forselectively killing senescent cells is RG-7112 (Roche) (CAS No:939981-39-2; IUPAC name:((4S,5R)-2-(4-(tert-butyl)-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethyl-4,5-dihydro-1H-imidazol-1-yl)(4-(3-(methylsulfonyl)propyl)piperazin-1-yl)methanone.See U.S. Pat. No. 7,851,626; Tovar et al., Cancer Res. 72:2587-97(2013).

In another particular embodiment, the MDM2 inhibitor is acis-imidazoline compound called RG7338 (Roche) (IPUAC Name:4-((2R,3S,4R,5S)-3-(3-chloro-2-fluorophenyl)-4-(4-chloro-2-fluorophenyl)-4-cyano-5-neopentylpyrrolidine-2-carboxamido)-3-methoxybenzoicacid) (CAS 1229705-06-9); Ding et al., J. Med. Chem. 56(14):5979-83.Doi: 10.1021/jm400487c. Epub 2013 Jul. 16; Zhao et al., J. Med. Chem.56(13):5553-61 (2013) doi: 10.1021/jm4005708. Epub 2013 Jun. 20). Yetanother exemplary nutlin compound is RO5503781. Other potentcis-imidazoline small molecule compounds include dihydroimidazothiazolecompounds (e.g., DS-3032b; Daiichi Sankyo) described by Miyazaki, (see,e.g., Miyazaki et al., Bioorg. Med. Chem. Lett. 23(3):728-32 (2013) doi:10.1016/j.bmcl.2012.11.091. Epub 2012 Dec. 1; Miyazaki et al., Bioorg.Med. Chem. Lett. 22(20):6338-42 (2012) doi: 10.1016/j.bmcl.2012.08.086.Epub 2012 Aug. 30; Int'l Patent Appl. Publ. No. WO 2009/151069 (2009)).

In still other embodiments, a cis-imidazoline compound that may be usedin the methods described herein is a dihydroimidazothiazole compound.

In other embodiments, the MDM2 small molecule inhibitor is aspiro-oxindole compound. See, for example, compounds described in Dinget al., J. Am. Chem. Soc. 2005; 127:10130-31; Shangary et al., Proc NatlAcad Sci USA 2008; 105:3933-38; Shangary et al., Mol Cancer Ther 2008;7:1533-42; Shangary et al., Mol Cancer Ther 2008; 7:1533-42; Hardcastleet al., Bioorg. Med. Chem. Lett. 15:1515-20 (2005); Hardcastle et al.,J. Med. Chem. 49(21):6209-21 (2006); Watson et al., Bioorg. Med. Chem.Lett. 21(19):5916-9 (2011) doi: 10.1016/j.bmcl.2011.07.084. Epub 2011Aug. 9. Other examples of spiro-oxindole compounds that are MDM2inhibitors are called in the art MI-63, MI-126; MI-122, MI-142, MI-147,MI-18, MI-219, MI-220, MI-221, and MI-773. Another specificspiro-oxindole compound is3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one.Another compound is called MI888 (see, e.g., Zhao et al., J. Med. Chem.56(13):5553-61 (2013); Int'l Patent Appl. Publ. No. WO 2012/065022).

In still other embodiments, the MDM2 small molecule inhibitor that maybe used in the methods described herein is a benzodiazepinedione (see,e.g., Grasberger et al., J Med Chem 2005; 48:909-12; Parks et al.,Bioorg Med Chem Lett 2005; 15:765-70; Raboisson et al., Bioorg. Med.Chem. Lett. 15:1857-61 (2005); Koblish et al., Mol. Cancer Ther.5:160-69 (2006)). Benzodiazepinedione compounds that may be used in themethods described herein include 1,4-benzodiazepin-2,5-dione compounds.Examples of benzodiazepinedione compounds include5-[(3S)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-7-phenyl-1,4-diazepin-1-yl]valericacid and5-[(3S)-7-(2-bromophenyl)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-yl]valericacid (see, e.g., Raboisson et al., supra). Other benzodiazepinedionecompounds are called in the art TDP521252 (IUPAC Name:5-[(3S)-3-(4-chlorophenyl)-4-[(1R)-1-(4-chlorophenyl)ethyl]-7-ethynyl-2,5-dioxo-3H-1,4-benzodiazepin-1-yl]pentanoicacid) and TDP665759 (IUPAC Name:(3S)-4-[(1R)-1-(2-amino-4-chlorophenyl)ethyl]-3-(4-chlorophenyl)-7-iodo-1-[3-(4-methylpiperazin-1-yl)propyl]-3H-1,4-benzodiazepine-2,5-dione)(see, e.g., Parks et al., supra; Koblish et al., supra) (Johnson &Johnson, New Brunswick, N.J.).

In yet another embodiment, the MDM2 small molecule inhibitor is aterphenyl (see, e.g., Yin et al., Angew Chem Int Ed Engi 2005;44:2704-707; Chen et al., Mol Cancer Ther 2005; 4:1019-25). In yetanother specific embodiment, the MDM2 inhibitor that may be used in themethods described herein is a quilinol (see, e.g., Lu et al., J Med Chem2006; 49:3759-62). In yet another certain embodiment, the MDM2 inhibitoris a chalcone (see, e.g., Stoll et al., Biochemistry 2001; 40:336-44).In yet another particular embodiment, the MDM2 inhibitor is asulfonamide (e.g., NSC279287) (see, e.g., Galatin et al., J Med Chem2004; 47:4163-65).

In other embodiments, a compound that may be used in the methodsdescribed herein is a tryptamine, such as serdemetan (JNJ-26854165;chemical name:N1-(2-(1H-indol-3-yl)ethyl)-N4-(pyridine-4-yl)benzene-1,4-diamine; CASNo. 881202-45-5) (Johnson & Johnson, New Brunswick, N.J.). Serdemetan isa tryptamine derivative that activates p53 and acts as a HDM2 ubiquitinligase antagonist (see, e.g., Chargari et al., Cancer Lett.312(2):209-18 (2011) doi: 10.1016/j.canlet.2011.08.011. Epub 2011 Aug.22; Kojima et al., Mol. Cancer Ther. 9:2545-57 (2010); Yuan et al., J.Hematol. Oncol. 4:16 (2011)).

In other particular embodiments, MDM2 small molecule inhibitors that maybe used in the methods described herein include those described in Rewet al., J. Med. Chem. 55:4936-54 (2012); Gonzalez-Lopez de Turiso etal., J. Med. Chem. 56:4053-70 (2013); Sun et al., J. Med. Chem.57:1454-72 (2014); Gonzalez et al., J. Med. Chem. 2014 Mar. 4 [Epubahead of print]; Gonzalez et al., J. Med. Chem. 2014 Mar. 6 [Epub aheadof print].

In still other embodiments, the MDM2 inhibitor is a piperidinonecompound. An example of a potent MDM2 piperidinone inhibitor is AM-8553({(3R,5R,6S)-5-(3-Chlorophenyl)-6-(4-chlorophenyl)-1-[(2S,3S)-2-hydroxy-3-pentanyl]-3-methyl-2-oxo-3-piperidinyl}aceticacid; CAS No. 1352064-70-0) (Amgen, Thousand Oaks, Calif.).

In other particular embodiments, an MDM2 inhibitor that may be used inthe methods described herein is a piperidine (Merck, Whitehouse Station,N.J.) (see, e.g., Int'l Patent Appl. Publ. No. WO 2011/046771). In otherembodiments, an MDM2 inhibitor that may be used in the methods is animidazole-indole compound (Novartis) (see, e.g., Int'l Patent Appl.Publ. No. WO 2008/119741).

Examples of compounds that bind to MDM2 and to MDMX and that may be usedin the methods described herein include RO-2443 and RO-5963((Z)-2-(4-((6-Chloro-7-methyl-1H-indol-3-yl)methylene)-2,5-dioxoimidazolidin-1-yl)-2-(3,4-difluorophenyl)-N-(1,3-dihydroxypropan-2-yl)acetamide)(see, e.g., Graves et al., Proc. Natl. Acad. Sci. USA 109:11788-93(2012); see also, e.g., Zhao et al., 2013, BioDiscovery, supra).

In another specific embodiment, an MDM2 inhibitor referred to in the artas CGM097 may be used in the methods described herein for selectivelykilling senescent cells and for treating a senescence-associated diseaseor disorder.

Inhibitors of BCL-2 Anti-Apoptotic Family of Proteins

In certain embodiments, the senolytic agent may be an inhibitor of oneor more proteins in the BCL-2 family. In certain embodiments, the atleast one senolytic agent is selected from an inhibitor of one or moreBCL-2 anti-apoptotic protein family members wherein the inhibitorinhibits at least BCL-xL. Inhibitors of BCL-2 anti-apoptotic family ofproteins alter at least a cell survival pathway. Apoptosis activationmay occur via an extrinsic pathway triggered by the activation of cellsurface death receptors or an intrinsic pathway triggered bydevelopmental cues and diverse intracellular stresses. This intrinsicpathway, also known as the stress pathway or mitochondrial pathway, isprimarily regulated by the BCL-2 family, a class of key regulators ofcaspase activation consisting of anti-apoptotic (pro-survival) proteinshaving BH1-BH4 domains (BCL-2 (i.e., the BCL-2 protein member of theBCL-2 anti-apoptotic protein family), BCL-xL, BCL-w, A1, MCL-1, andBCL-B); pro-apoptotic proteins having BH1, BH2, and BH3 domains (BAX,BAK, and BOK); and pro-apoptotic BH3-only proteins (BIK, BAD, BID, BIM,BMF, HRK, NOXA, and PUMA) (see, e.g., Cory et al., Nature Reviews Cancer2:647-56 (2002); Cory et al., Cancer Cell 8:5-6 (2005); Adams et al.,Oncogene 26:1324-1337 (2007)). BCL-2 anti-apoptotic proteins blockactivation of pro-apoptotic multi-domain proteins BAX and BAK (see,e.g., Adams et al., Oncogene 26:1324-37 (2007)). While the exactmechanism of apoptosis regulation is unknown, it is hypothesized thatBH3-only proteins unleashed by intracellular stress signals bind toanti-apoptotic BCL-2 like proteins via a BH3 “ligand” to a “receptor”BH3 binding groove formed by BH1-3 regions on anti-apoptotic proteins,thereby neutralizing the anti-apoptotic proteins (see, e.g., Letai etal., Cancer Cell 2:183-92 (2002); Adams et al., Oncogene, supra). BAXand BAK can then form oligomers in mitochondrial membranes, leading tomembrane permeabilization, release of cytochrome C, caspase activation,and ultimately apoptosis (see, e.g., Adams et al., Oncogene, supra).

As used herein and unless otherwise stated, a BCL-2 family member thatis inhibited by the agents described herein is a pro-survival(anti-apoptotic) family member. The senolytic agents used in the methodsdescribed herein inhibit one or more functions of the BCL-2anti-apoptotic protein, BCL-xL (which may also be written herein and inthe art as Bcl-xL, BCL-XL, Bcl-xl, or Bcl-XL). In certain embodiments,in addition to inhibiting BCL-xL function, the inhibitor may alsointeract with and/or inhibit one or more functions of BCL-2 (i.e.,BCL-xL/BCL-2 inhibitors). In yet another certain embodiment, senolyticagents used in the methods described herein are classified as inhibitorsof each of BCL-xL and BCL-w (i.e., BCL-xL/BCL-w inhibitors). In stillanother specific embodiment, senolytic agents used in the methodsdescribed herein that inhibit BCL-xL may also interact with and inhibitone or more functions of each of BCL-2 (i.e., the BCL-2 protein) andBCL-w (i.e., BCL-xL/BCL-2/BCL-w inhibitors), thereby causing selectivekilling of senescent cells. In certain embodiments, a BCL-2anti-apoptotic protein inhibitor interferes with the interaction betweenthe BCL-2 anti-apoptotic protein family member (which includes at leastBCL-xL) and one or more ligands or receptors to which the BCL-2anti-apoptotic protein family member would bind in the absence of theinhibitor. In other particular embodiments, an inhibitor of one or moreBCL-2 anti-apoptotic protein family members wherein the inhibitorinhibits at least BCL-xL specifically binds only to one or more ofBCL-xL, BCL-2, BCL-w and not to other Bcl-2 anti-apoptotic Bcl-2 familymembers, such as Mcl-1 and BCL2A1.

In still another embodiment, the senolytic agent used in the methodsdescribed herein is a BCL-xL selective inhibitor and inhibits one ormore functions of BCL-xL. Such senolytic agents that are BCL-xLselective inhibitors do not inhibit the function of one or more otherBCL-2 anti-apoptotic proteins in a biologically or statisticallysignificant manner. BCL-xL may also be called BCL2L1, BCL2-like 1, BCLX,BCL2L, BCLxL, or BCL-X herein and in the art. In one embodiment, BCL-xLselective inhibitors alter (e.g., reduce, inhibit, decrease, suppress)one or more functions of BCL-xL but do not significantly inhibit one ormore functions of other proteins in the BCL-2 anti-apoptotic proteinfamily (e.g., BCL-2 or BCL-w). In certain embodiments, a BCL-xLselective inhibitor interferes with the interaction between BCL-xL andone or more ligands or receptors to which BCL-xL would bind in theabsence of the inhibitor. In certain particular embodiments, a senolyticagent that inhibits one or more of the functions of BCL-xL selectivelybinds to human BCL-xL but not to other proteins in the BCL-2 family,which effects selective killing of senescent cells.

BCL-xL is an anti-apoptotic member of the BCL-2 protein family. BCL-xLalso plays an important role in the crosstalk between autophagy andapoptosis (see, e.g., Zhou et al., FEBS J. 278:403-13 (2011)). BCL-xLalso appears to play a role in bioenergetic metabolism, includingmitochondrial ATP production, Ca²⁺ fluxes, and protein acetylation, aswell as on several other cellular and organismal processes such asmitosis, platelet aggregation, and synaptic efficiency (see, e.g.,Michels et al., International Journal of Cell Biology, vol. 2013,Article ID 705294, 10 pages, 2013. doi:10.1155/2013/705294). In certainembodiments, the BCL-xL inhibitors described herein may disrupt theinteraction between BCL-xL and any one or more of the aforementionedBH3-only proteins to promote apoptosis in cells.

In certain embodiments, a BCL-xL inhibitor is a selective inhibitor,meaning, that it preferentially binds to BCL-xL over otheranti-apoptotic BCL2 family members (e.g., BCL-2, MCL-1, BCL-w, BCL-b,and BFL-1/A1). In certain embodiments, a BCL-XL selective inhibitorexhibits at least a 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold,10000-fold, 20000-fold, or 30000-fold selectivity for binding a BCL-XLprotein or nucleic acid over a BCL-2 protein or nucleic acid. In certainembodiments, a BCL-xL selective inhibitor exhibits at least a 5-fold,10-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 20000-fold, or30000-fold selectivity for binding a BCL-xL protein or nucleic acid overa MCL-1 protein or nucleic acid. In certain embodiments, a BCL-xLselective inhibitor exhibits at least a 5-fold, 10-fold, 50-fold,100-fold, 1000-fold, 10000-fold, 20000-fold, or 30000-fold selectivityfor binding a BCL-xL protein or nucleic acid over a BCL-w protein ornucleic acid. In certain embodiments, a BCL-xL selective inhibitorexhibits at least a 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold,10000-fold, 20000-fold, or 30000-fold selectivity for binding a BCL-XLprotein or nucleic acid over a BCL-B protein or nucleic acid. In certainembodiments, a BCL-XL selective inhibitor exhibits at least a 5-fold,10-fold, 50-fold, 100-fold, 1000-fold, 10000-fold, 20000-fold, or30000-fold selectivity for binding a BCL-xL protein or nucleic acid overan A1 protein or nucleic acid. As described herein, in certainembodiments, an inhibitor of one or more BCL-2 anti-apoptotic proteinfamily members wherein the inhibitor inhibits at least BCL-xL (e.g., aBCL-xL selective inhibitor) has no detectable binding to MCL-1 or toBCL2A1.

Methods for measuring binding affinity of a BCL-xL inhibitor for BCL-2family proteins are known in the art. By way of example, bindingaffinity of a BCL-xL inhibitor may be determined using a competitionfluorescence polarization assay in which a fluorescent BAK BH3 domainpeptide is incubated with BCL-xL protein (or other BCL-2 family protein)in the presence or absence of increasing concentrations of the BCL-XLinhibitor as previously described (see, e.g., U.S. Patent Publication20140005190; Park et al., Cancer Res. 73:5485-96 (2013); Wang et al.,Proc. Natl. Acad. Sci USA 97:7124-9 (2000); Zhang et al., Anal. Biochem.307:70-5 (2002); Bruncko et al., J. Med. Chem. 50:641-62 (2007)).Percent inhibition may be determined by the equation: 1-[(mP value ofwell−negative control)/range)]×100%. Inhibitory constant (K_(i)) valueis determined by the formula: K_(i)=[I]₅₀/([L]₅₀/K_(d)+[P]₀/K_(d)+1) asdescribed in Bruncko et al., J. Med. Chem. 50:641-62 (2007) (see, also,Wang, FEBS Lett. 360:111-114 (1995)).

Agents (e.g., BCL-xL selective inhibitors, BCL-xL/BCL-2 inhibitors,BCL-xL/BCL-2/BCL-w inhibitors, BCL-xL/BCL-w inhibitors) used in themethods described herein that selectively kill senescent cells include,by way of example, a small molecule.

In particular embodiments, the BCL-xL inhibitor is a small moleculecompound that belongs to any one of the following classes of compounds,for example, a benzothiazole-hydrazone compound, aminopyridine compound,benzimidazole compound, tetrahydroquinoline compound, and phenoxylcompound and related analogs.

In one embodiment, a BCL-xL selective inhibitor useful for the methodsdescribed herein is a benzothiazole-hydrazone small molecule inhibitor.Benzothiazole-hydrazone compounds include WEHI-539(5-[3-[4-(aminomethyl)phenoxy]propyl]-2-[(8E)-8-(1,3-benzothiazol-2-ylhydrazinylidene)-6,7-dihydro-5H-naphthalen-2-yl]-1,3-thiazole-4-carboxylicacid), a BH3 peptide mimetic that selectively targets BCL-xL (see, e.g.,Lessene et al., Nature Chemical Biology 9:390-397 (2013)). In certainembodiments, the methods described herein comprise use of WEHI-539 forselectively killing senescent cells.

In other embodiments, the BCL-xL selective inhibitor is an aminopyridinecompound. An aminopyridine compound that may be used as a selectiveBCL-xL inhibitor is BXI-61(3-[(9-amino-7-ethoxyacridin-3-yl)diazenyl]pyridine-2,6-diamine) (see,e.g., Park et al., Cancer Res. 73:5485-96 (2013); U.S. Patent Publ. No.2009-0118135). In certain embodiments, the methods described hereincomprise use of BXI-61 for selectively killing senescent cells.

In still other embodiments, the BCL-xL selective inhibitor that may beused in the methods described herein is a benzimidazole compound. Anexample of a benzimidazole compound that may be used as a selectiveBCL-XL inhibitor is BXI-72(2′-(4-Hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi(1H-benzimidazole)trihydrochloride) (see, e.g., Park et al., supra). In certainembodiments, the methods described herein comprise use of BXI-72 forselectively killing senescent cells.

In yet another embodiment, the BCL-xL selective inhibitor is atetrahydroquinoline compound (see, e.g., U.S. Patent Publ. No.2014-0005190). Examples of tetrahydroquinoline compounds that may beused as selective BCL-xL inhibitors are shown in Table 1 of U.S. PatentPubl. No. 2014-0005190 and described therein. Other inhibitors describedtherein may inhibit other BCL-2 family members (e.g., BCL-2) in additionto BCL-xL.

In other embodiments, a BCL-xL selective inhibitor is a phenoxylcompound. An example of a phenoxyl compound that may be used as aselective BCL-xL inhibitor is 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol (2,3-DCPE) (see, Wu et al., Cancer Res.64:1110-1113 (2004)). In certain embodiments, the methods describedherein comprise use of 2,3-DCPE for selectively killing senescent cells.

In still another embodiment, an inhibitor of a Bcl-2 anti-apoptoticfamily member that inhibits at least BCL-xL is described in U.S. Pat.No. 8,232,273. In a particular embodiment, the inhibitor is a BCL-xLselective inhibitor called A-1155463 (see, e.g., Tao et al., ACS Med.Chem. Lett., 2014, 5(10): 1088-1093).

In other embodiments, a senolytic agent of interest inhibits other BCL-2anti-apoptotic family members in addition to BCL-xL. For example,methods described herein comprise use of BCL-xL/BCL-2 inhibitors,BCL-xL/BCL-2/BCL-w inhibitors, and BCL-xL/BCL-w inhibitors and analogsthereof. In certain embodiments, the inhibitors include compounds thatinhibit BCL-2 and BCL-xL, which inhibitors may also inhibit BCL-w.Examples of these inhibitors include ABT-263(4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamideor IUPAC,(R)-4-(4-((4′-chloro-4,4-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholino-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)phenyl)sulfonyl)benzamide)(see, e.g., Park et al., 2008, J. Med. Chem. 51:6902; Tse et al., CancerRes., 2008, 68:3421; Int'l Patent Appl. Pub. No. WO 2009/155386; U.S.Pat. Nos. 7,390,799, 7,709,467, 7,906,505, 8,624,027) and ABT-737(4-[4-[(4′-Chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]benzamide,Benzamide,4-[4-[(4′-chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-or4-[4-[[2-(4-chlorophenyl)phenyl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-(dimethylamino)-1-phenylsulfanylbutan-2-yl]amino]-3-nitrophenyl]sulfonylbenzamide)(see, e.g., Oltersdorf et al., Nature, 2005, 435:677; U.S. Pat. No.7,973,161; U.S. Pat. No. 7,642,260). In other embodiments, the BCL-2anti-apoptotic protein inhibitor is a quinazoline sulfonamide compound(see, e.g., Sleebs et al., 2011, J. Med. Chem. 54:1914). In stillanother embodiment, the BCL-2 anti-apoptotic protein inhibitor is asmall molecule compound as described in Zhou et al., J. Med. Chem.,2012, 55:4664 (see, e.g., Compound 21(R)-4-(4-chlorophenyl)-3-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-5-ethyl-1-methyl-1H-pyrrole-2-carboxylicacid) and Zhou et al., J. Med. Chem., 2012, 55:6149 (see, e.g., Compound14(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-ethyl-2-methyl-1H-pyrrole-3-carboxylicacid; Compound 15(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-((4-(dimethylamino)-1-(phenylthio)butan-2-yl)amino)-3-nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylicacid). In other embodiments, the BCL-2 anti-apoptotic protein inhibitoris a BCL-2/BCL-xL inhibitor such as BM-1074 (see, e.g., Aguilar et al.,2013, J. Med. Chem. 56:3048); BM-957 (see, e.g., Chen et al., 2012, J.Med. Chem. 55:8502); BM-1197 (see, e.g., Bai et al., PLoS One 2014 Jun.5; 9(6):e99404. Doi: 10.1371/journal.pone. 009904); U.S. Patent Appl.No. 2014/0199234; N-acylsulfonamide compounds (see, e.g., Int'l PatentAppl. Pub. No. WO 2002/024636, Int'l Patent Appl. Pub. No. WO2005/049593, Int'l Patent Appl. Pub. No. WO 2005/049594, U.S. Pat. No.7,767,684, U.S. Pat. No. 7,906,505). In still another embodiment, theBCL-2 anti-apoptotic protein inhibitor is a small molecule macrocycliccompound (see, e.g., Int'l Patent Appl. Pub. No. WO 2006/127364, U.S.Pat. No. 7,777,076). In yet another embodiment, the BCL-2 anti-apoptoticprotein inhibitor is an isoxazolidine compound (see, e.g., Int'l PatentAppl. Pub. No. WO 2008/060569, U.S. Pat. No. 7,851,637, U.S. Pat. No.7,842,815).

In certain embodiments, the senolytic agent is a compound that is aninhibitor of Bcl-2, Bcl-w, and Bcl-xL, such as ABT-263 or ABT-737. Incertain specific embodiments, the senolytic agent is a compound or apharmaceutically acceptable salt, stereoisomer, tautomer, or prodrugthereof as illustrated below, which depicts the structure of ABT-263.ABT-263 is also known as Navitoclax in the art.

Akt Kinase Inhibitors

In certain embodiments the senolytic agent is an Akt Kinase inhibitor.For example, a senolytic agent can be a small molecule compound andanalogs thereof that inhibits Akt. In some embodiments, the senolyticagent is a compound that selectively inhibits Akt1, Akt2, and Akt3,relative to other protein kinases.

Akt inhibitors (which may also be called Akt kinase inhibitors or AKTkinase inhibitors) can be divided into six major classes based on theirmechanisms of action (see, e.g., Bhutani et al., Infectious Agents andCancer 2013, 8:49 doi:10.1186/1750-9378-8-49). Akt is also calledprotein kinase B (PKB) in the art. The first class contains ATPcompetitive inhibitors of Akt and includes compounds such as CCT128930and GDC-0068, which inhibit Akt2 and Akt1. This category also includesthe pan-Akt kinase inhibitors such as GSK2110183 (afuresertib),GSK690693, and AT7867. The second class contains lipid-based Aktinhibitors that act by inhibiting the generation of PIP3 by PI3K. Thismechanism is employed by phosphatidylinositol analogs such as CalbiochemAkt Inhibitors I, II and III or other PI3K inhibitors such as PX-866.This category also includes compounds such as Perifosine (KRX-0401)(Aeterna Zentaris/Keryx). The third class contains a group of compoundscalled pseudosubstrate inhibitors. These include compounds such asAKTide-2 T and FOXO3 hybrid. The fourth class consists of allostericinhibitors of AKT kinase domain, and include compounds such as MK-2206(8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazol[3,4f][1,6]naphthyridin-3-one;dihydrochloride) (Merck & Co.) (see, e.g., U.S. Pat. No. 7,576,209). Thefifth class consists of antibodies and include molecules such asGST-anti-Akt1-MTS. The last class comprises compounds that interact withthe PH domain of Akt, and includes Triciribine and PX-316. Othercompounds described in the art that act as AKT inhibitors include, forexample, GSK-2141795 (GlaxoSmithKline), VQD-002, miltefosine, AZD5363,GDC-0068, and API-1. Techniques for determining the activity of AKTinhibitors are routinely practiced by persons skilled in the art

In a specific embodiment, the senolytic agent is a compound that is anAkt kinase inhibitor, which has the structure as shown below (alsocalled MK-2206 herein and in the art),8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-2H-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3-one;)or a pharmaceutically acceptable salt, stereoisomer, tautomer, orprodrug thereof. The dihydrochloride salt is shown.

In certain embodiments, at least one senolytic agent may be administeredwith at least one other senolytic agent, which two or more senolyticagents act additively or synergistically to selectively kill senescentcells. In particular embodiments, methods are provided for using asenolytic agent wherein the senolytic agent alters either a cellsurvival signaling pathway or an inflammatory pathway or alters both thecell survival signaling pathway and the inflammatory pathway in asenescent cell. In other particular embodiments, methods comprise use ofat least two senolytic agents wherein at least one senolytic agent and asecond senolytic agent are each different and independently alter eitherone or both of a survival signaling pathway and an inflammatory pathwayin a senescent cell. For convenience, when two or more senolytic agentsare described herein as being used in combination, one senolytic agentwill be called a first senolytic agent, another senolytic agent will becalled the second senolytic agent, etc. In other certain embodiments,the methods described herein comprise administering at least threesenolytic agents (a first senolytic agent, second senolytic agent, andthird senolytic agent). The adjectives, first, second, third, and such,in this context are used for convenience only and are not to beconstrued as describing order or administration, preference, or level ofsenolytic activity or other parameter unless expressly describedotherwise. In particular embodiments, when two or more senolytic agentsare used in the methods described herein, each senolytic agent is asmall molecule. In other certain embodiments, the methods describedherein comprise administering at least three senolytic agents (a firstsenolytic agent, second senolytic agent, and third senolytic agent). Incertain embodiments, use of at least two senolytic agents results insignificantly increased killing of senescent cells compared with use ofeach senolytic agent alone. In other particular embodiments, use of atleast two senolytic agents results in significant killing of senescentcells compared with use of each senolytic agent alone and which effectmay be additive or synergistic. In certain embodiments, the at least twosenolytic agents are each different and selected from (1) an inhibitorof one or more BCL-2 anti-apoptotic protein family members wherein theinhibitor inhibits at least BCL-xL; (for example, a Bcl-2/Bcl-xL/Bcl-winhibitor, a Bcl-2/Bcl-xL inhibitor, a selective Bcl-xL inhibitor, or aBcl-xL/Bcl-w inhibitor); an Akt kinase specific inhibitor; a MDM2inhibitor. In one particular embodiment, when at least one senolyticagent administered to a subject in need thereof is an inhibitor of oneor more BCL-2 anti-apoptotic protein family members wherein theinhibitor inhibits at least BCL-XL (e.g., a Bcl-2/Bcl-xL/Bcl-winhibitor, a Bcl-2/Bcl-xL inhibitor, a selective Bcl-xL inhibitor, or aBcl-xL/Bcl-w inhibitor), a second senolytic agent is administered. Inother certain embodiments, one of the two senolytic agents is theinhibitor of one or more BCL-2 anti-apoptotic protein family memberswherein the inhibitor inhibits at least BCL-xL and the second senolyticagent is an MDM2 inhibitor. In yet still more particular embodiments,when at least one senolytic agent administered to a subject in needthereof is a selective Bcl-xL inhibitor, a second senolytic agent isadministered. In still more particular embodiments, when at least onesenolytic agent administered to a subject in need thereof is an MDM2inhibitor, a second senolytic agent is administered. In still moreparticular embodiments, when at least one senolytic agent administeredto a subject in need thereof is an Akt kinase inhibitor, a secondsenolytic agent is administered. In even more particular embodiments,the inhibitor of one or more BCL-2 anti-apoptotic protein family memberswherein the inhibitor inhibits at least BCL-xL is used alone or incombination with another senolytic agent that is also an inhibitor ofone or more BCL-2 anti-apoptotic protein family members wherein theinhibitor inhibits at least BCL-xL or is a different senolytic agent asdescribed herein. In particular embodiments, an inhibitor of one or moreBCL-2 anti-apoptotic protein family members wherein the inhibitorinhibits at least BCL-xL is combined with an inhibitor of Akt kinase. Byway of non-limiting example, the Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-263may be used in combination with an Akt kinase inhibitor (e.g., MK2206).

In still other particular embodiments, an MDM2 inhibitor that is asenolytic agent is used in combination with at least one additionalsenolytic agent in the methods for treating a senescence-associateddisease or disorder; the additional senolytic agent (which may bereferred to for convenience as a second senolytic agent) may be anotherMDM2 inhibitor or may be a senolytic agent that is not a MDM2 inhibitor.In one embodiment, an inhibitor of a Bcl-2 anti-apoptotic family memberthat inhibits at least Bcl-xL is used in combination with an AKTinhibitor. In a more specific embodiment, the inhibitor of a Bcl-2anti-apoptotic family member is ABT-263, ABT-737, or WEHI-539 and theAKT inhibitor is MK-2206.

In other certain embodiments, the methods described herein compriseadministering at least three senolytic agents (a first senolytic agent,second senolytic agent, and third senolytic agent).

mTOR, NFκB, and PI3-k Pathway Inhibitors: A small molecule compound thatmay be used together with a senolytic agent described herein in themethods for selectively killing senescent cells and treating asenescence-associated disease or disorder may be a small moleculecompound that inhibits one or more of mTOR, NFκB, and PI3-k pathways. Asdescribed herein, methods are also provided for selectively killingsenescent cells and for treating a senescence-associated disease ordisorder, wherein the methods comprise administering to a subject inneed thereof at least one senolytic agent, which methods may furthercomprise administering an inhibitor of one or more of mTOR, NFκB, andPI3-k pathways. Inhibitors of these pathways are known in the art.

Examples of mTOR inhibitors include sirolimus, temsirolimus, everolimus,ridaforolimus, 32-deoxorapamycin, zotarolimus, PP242, INK128, PP30,Torin1, Ku-0063794, WAY-600, WYE-687 and WYE-354. Inhibitors of an NFκBpathway include, for example, NFκB activity abrogation through TPCA-1(an IKK2 inhibitor); BAY 11-7082 (an IKK inhibitor poorly selective forIKK1 and IKK2); and MLN4924 (an NEDD8 activating enzyme(NAE)-inhibitor); and MG132.

Examples of inhibitors of PI3k that may also inhibit mTOR or AKTpathways include perifosine (KRX-0401), idelalisib, PX-866, IPI-145, BAY80-6946, BEZ235, RP6530, TGR 1201, SF1126, INK1117, GDC-0941, BKM120,XL147 (SAR245408), XL765 (SAR245409), Palomid 529, GSK1059615,GSK690693, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, RP6503,PI-103, GNE-477, CUDC-907, AEZS-136, BYL719, BKM120, GDC-0980, GDC-0032,and MK2206.

Small Molecule Compounds—Salts and General Synthesis Procedures.

The small molecule compounds described herein as senolytic agentsinclude physiologically acceptable salts (i.e., pharmaceuticallyacceptable salts), hydrates, solvates, polymorphs, metabolites, andprodrugs of the senolytic agents. Further information on metabolism maybe obtained from The Pharmacological Basis of Therapeutics, 9th Edition,McGraw-Hill (1996). Metabolites of the compounds disclosed herein can beidentified either by administration of compounds to a host and analysisof tissue samples from the host, or by incubation of compounds withhepatic cells in vitro and analysis of the resulting compounds. Bothmethods are well known in the art.

The compounds described herein may generally be used as the free acid orfree base. Alternatively, the compounds may be used in the form of acidor base addition salts. Acid addition salts of the free base aminocompounds may be prepared according to methods well known in the art,and may be formed from organic and inorganic acids. Suitable organicacids include (but are not limited to) maleic, fumaric, benzoic,ascorbic, succinic, methanesulfonic, acetic, oxalic, propionic,tartaric, salicylic, citric, gluconic, lactic, mandelic, cinnamic,aspartic, stearic, palmitic, glycolic, glutamic, malonic, andbenzenesulfonic acids. Suitable inorganic acids include (but are notlimited to) hydrochloric, hydrobromic, sulfuric, phosphoric, and nitricacids. Base addition salts of the free acid compounds of the compoundsdescribed herein may also be prepared by methods well known in the art,and may be formed from organic and inorganic bases. Additional saltsinclude those in which the counterion is a cation. Suitable inorganicbases included (but are not limited to) the hydroxide or other salt ofsodium, potassium, lithium, ammonium, calcium, barium, magnesium, iron,zinc, copper, manganese, aluminum, and the like, and organic bases suchas substituted ammonium salts (for example, dibenzylammonium,benzylammonium, 2-hydroxyethylammonium). Further salts include those inwhich the counterion is an anion, such as adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.Thus, the term “pharmaceutically acceptable salt” of compounds describedherein is intended to encompass any and all pharmaceutically suitablesalt forms.

Compounds may sometimes be depicted as an anionic species. One ofordinary skill in the art will recognize that the compounds exist withan equimolar ratio of cation. For instance, the compounds describedherein can exist in the fully protonated form, or in the form of a saltsuch as sodium, potassium, ammonium or in combination with any inorganicbase as described above. When more than one anionic species is depicted,each anionic species may independently exist as either the protonatedspecies or as the salt species. In some specific embodiments, thecompounds described herein exist as the sodium salt. In other specificembodiments, the compounds described herein exist as the potassium salt.

Furthermore, some of the crystalline forms of any compound describedherein may exist as polymorphs, which are also included and contemplatedby the present disclosure. In addition, some of the compounds may formsolvates with water or other organic solvents. Often crystallizationsproduce a solvate of the disclosed compounds. As used herein, the term“solvate” refers to an aggregate that comprises one or more molecules ofany of the disclosed compounds with one or more molecules of solvent.The solvent may be water, in which case the solvate may be a hydrate.Alternatively, the solvent may be an organic solvent. Thus, thepresently disclosed compounds may exist as a hydrate, including amonohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate,tetrahydrate and the like, as well as the corresponding solvated forms.Certain embodiments of the compounds may be true solvates, while inother instances, some embodiments of the compounds may merely retainadventitious water or be a mixture of water plus some adventitioussolvent.

In general, the compounds used in the methods described herein may bemade according to organic synthesis techniques known to those skilled inthis art, starting from commercially available chemicals and/or fromcompounds described in the chemical literature. Specific and analogousreactants may also be identified through the indices of known chemicalsprepared by the Chemical Abstract Service of the American ChemicalSociety, which are available in most public and university libraries, aswell as through on-line databases (the American Chemical Society,Washington, D.C., may be contacted for more details). Chemicals that areknown but not commercially available in catalogs may be prepared bycustom chemical synthesis houses, where many of the standard chemicalsupply houses (e.g., those listed above) provide custom synthesisservices. A reference for the preparation and selection ofpharmaceutical salts of the present disclosure is P. H. Stahl & C. G.Wermuth “Handbook of Pharmaceutical Salts,” Verlag Helvetica ChimicaActa, Zurich, 2002. Methods known to one of ordinary skill in the artmay be identified through various reference books and databases.Suitable reference books and treatises detail the synthesis of reactantsuseful in the preparation of compounds described herein, or providereferences to articles that describe the preparation.

Assays and techniques for identifying senolytic agents are described ingreater detail herein. In addition, identifying and selecting smallcompounds as senolytic agents, a person skilled in the medicinalchemistry art may also consider other properties of the small molecule,such as solubility, bioavailability, pharmacokinetics, Lipinski Rule of5, and the like.

Polypeptides, Antibodies, and Nucleic Acids

In other certain embodiments, a senolytic agent may be a polypeptide,peptide, antibody, antigen-binding fragment (i.e., peptides andpolypeptides comprising at least one complementary determining region(CDR)), peptibody, recombinant viral vector, or a nucleic acid. Incertain embodiments, a senolytic agent is an antisense oligonucleotide,siRNA, shRNA, or a peptide. For example, senolytic agents such aspolypeptides, antibodies, nucleic acids, and the like, include, forexample, MDM2 inhibitors, BCL-2 family inhibitors, or Akt kinaseinhibitors. In other embodiments, polypeptides, peptides, antibodies(including antigen-binding fragments thereof) that specifically bind toa ligand or target protein of a small molecule senolytic agent describedherein, may be used in assays and methods for characterizing ormonitoring the use of the small molecule senolytic agent.

A polynucleotide or oligonucleotide that specifically hybridizes to aportion of mRNA that encodes a target protein (e.g., Bcl-xL, Bcl-2,Bcl-w, MDM2, Akt) of a cell that is a senescent cell or that is a cellin a disease microenvironment may induce the cell to senescence byaging, a biologically damaging (i.e., cell damaging) medical therapy, oran environmental insult. In other embodiments, the target protein may bea ligand, or protein either downstream or upstream in a cell survivalpathway or inflammatory pathway or apoptotic pathway. Polynucleotidesand oligonucleotides may be complementary to at least a portion of anucleotide sequence encoding a target polypeptide (e.g., a shortinterfering nucleic acid, an antisense polynucleotide, a ribozyme, or apeptide nucleic acid) and that may be used to alter gene and/or proteinexpression. These polynucleotides that specifically bind to or hybridizeto nucleic acid molecules that encode a target polypeptide may beprepared using the nucleotide sequences available in the art. In anotherembodiment, nucleic acid molecules such as aptamers that are notsequence-specific may also be used to alter gene and/or proteinexpression.

Antisense polynucleotides bind in a sequence-specific manner to nucleicacids such as mRNA or DNA. Identification of oligonucleotides andribozymes for use as antisense agents and identification of DNA encodingthe target gene for targeted delivery involve methods well known in theart. For example, the desirable properties, lengths, and othercharacteristics of such oligonucleotides are well known. Antisensetechnology can be used to control gene expression through interferencewith binding of polymerases, transcription factors, or other regulatorymolecules (see, e.g., Gee et al., In Huber and Carr, Molecular andImmunologic Approaches, Futura Publishing Co. (Mt. Kisco, N.Y.; 1994)).

Short interfering RNAs may be used for modulating (decreasing orinhibiting) the expression of a gene encoding a target polypeptide ofinterest (see, e.g., Examples herein). Small nucleic acid molecules,such as short interfering RNA (siRNA), micro-RNA (miRNA), and shorthairpin RNA (shRNA) molecules may be used according to the methodsdescribed herein to modulate the expression of a target protein. A siRNApolynucleotide preferably comprises a double-stranded RNA (dsRNA) butmay comprise a single-stranded RNA (see, e.g., Martinez et al., Cell110:563-74 (2002)). A siRNA polynucleotide may comprise other naturallyoccurring, recombinant, or synthetic single-stranded or double-strandedpolymers of nucleotides (ribonucleotides or deoxyribonucleotides or acombination of both) and/or nucleotide analogues as provided herein andknown and used by persons skilled in the art.

The term “siRNA” refers to a double-stranded interfering RNA unlessotherwise noted. Typically, an siRNA is a double-stranded nucleic acidmolecule comprising two nucleotide strands, each strand having about 19to about 28 nucleotides (i.e., about 19, 20, 21, 22, 23, 24, 25, 26, 27,or 28 nucleotides). In certain embodiments, each strand is 19, 20, 21,22, or 23 nucleotides. In other particular embodiments, the siRNAcomprises two nucleotide strands, each strand having about 15, 16, 17,or 18 nucleotides. In other certain embodiments, one strand of thedouble stranded siRNA is at least two nucleotides longer, for example,one strand may have a two-base overhang (such as TT) at one end, usuallythe 3′ terminal end.

Short hairpin interfering RNA molecules comprise both the sense andantisense strands of an interfering RNA in a stem-loop or hairpinstructure (e.g., a shRNA). An shRNA may be expressed from a DNA vectorin which the DNA oligonucleotides encoding a sense interfering RNAstrand are linked to the DNA oligonucleotides encoding the reversecomplementary antisense interfering RNA strand by a short spacer. Ifneeded, 3′ terminal T's and nucleotides forming restriction sites may beadded. The resulting RNA transcript folds back onto itself to form astem-loop structure.

In addition to siRNA molecules, other interfering RNA and RNA-likemolecules can interact with RISC and silence gene expression, such asshort hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs),and dicer-substrate 27-mer duplexes. Such RNA-like molecules may containone or more chemically modified nucleotides, one or morenon-nucleotides, one or more deoxyribonucleotides, and/or one or morenon-phosphodiester linkages. RNA or RNA-like molecules that can interactwith RISC and participate in RISC-related changes in gene expression maybe referred to herein as “interfering RNAs” or “interfering RNAmolecules.” Single-stranded interfering RNA in certain instances effectsmRNA silencing, but less efficiently than double-stranded RNA.

A person skilled in the art will also recognize that RNA molecules, suchas siRNA, miRNA, shRNA, may be chemically modified to confer increasedstability against nuclease degradation while retaining the capability tobind to the target nucleic acids that may be present in cells. The RNAmay be modified at any position of the molecule so long as the modifiedRNA binds to the target sequence of interest and resists enzymaticdegradation. Modifications in the siRNA may be in the nucleotide base,the ribose, or the phosphate. By way of example, the 2′ position ofribose can be modified, which modification can be accomplished using anyone of a number of different methods routinely practiced in the art. AnRNA may be chemically modified by the addition of a halide such asfluoro. Other chemical moieties that have been used to modify RNAmolecules include methyl, methoxyethyl, and propyl groups (see, e.g.,U.S. Pat. No. 8,675,704).

In a particular embodiment, the polynucleotide or oligonucleotide (e.g.,including a shRNA) may be delivered by a recombinant vector in which thepolynucleotide or oligonucleotide of interest has been incorporated. Inother embodiments, the recombinant viral vector may be a recombinantexpression vector into which a polynucleotide sequence that encodes anantibody, an antigen-binding fragment, polypeptide or peptide thatinhibits a protein in a cell survival pathway or an inflammatorypathway, including the proteins described herein such as Bcl-xL, Bcl-2,Bcl-w, MDM2, and Akt is inserted such that the encoding sequence isoperatively linked with one or more regulatory control sequences todrive expression of the polypeptide, antibody, an antigen-bindingfragment, or peptide. The recombinant vector or the recombinantexpression vector may be a viral recombinant vector or a viralrecombinant expression vector. Exemplary viral vectors include, withoutlimitation, a lentiviral vector genome, poxvirus vector genome, vacciniavirus vector genome, adenovirus vector genome, adenovirus-associatedvirus vector genome, herpes virus vector genome, and alpha virus vectorgenome. Viral vectors may be live, attenuated, replication conditionalor replication deficient, and typically is a non-pathogenic (defective),replication competent viral vector. Procedures and techniques fordesigning and producing such viral vectors are well known to androutinely practiced by persons skilled in the art.

In certain specific embodiments a senolytic agent that may be used inthe methods described herein is an antisense oligonucleotide. By way ofnon-limiting example, BCL-xL specific antisense oligonucleotides thathave been previously described may be used in the methods describedherein (see, e.g., PCT Publ. No. WO 00/66724; Xu et al., Intl. J. Cancer94:268-74 (2001); Olie et al., J. Invest. Dermatol. 118:505-512 (2002);and Wacheck et al., Br. J. Cancer 89:1352-1357 (2003)).

In certain embodiments, a senolytic agent that may be used in themethods described herein is a peptide. By way of example and in certainembodiments, a BCL-xL selective peptide inhibitor is a BH3 peptidemimetic. Examples of BCL-xL selective BH3 peptide mimetics include thosepreviously described (see, e.g., Kutzki et al., J. Am. Chem. Soc.124:11838-39 (2002); Yin et al., Bioorg. Med. Chem. Lett. 22:1375-79(2004); Matsumura et al., FASEB J. 7:2201 (2010)).

In certain embodiments, a senolytic agent useful for the methodsdescribed herein does not include a polynucleotide, or a fragmentthereof, that encodes the exonuclease, EXO1, or a vector (including aviral vector) that comprises a polynucleotide that encodes the EXO1enzyme (i.e., a polynucleotide encoding an EXO1 enzyme, a fragment ofthe polynucleotide, or a vector containing such a polynucleotide isexcluded). A senolytic agent useful for the methods described hereinalso does not include the EXO1 enzyme polypeptide (i.e., the EXO1 enzymeis excluded) or biologically active peptide or polypeptide fragmentthereof. In addition, such molecules are not inhibitors of one or bothof a cell signaling pathway, such as an inflammatory pathway or a cellsurvival pathway; instead EXO1 encodes a 5′-3′ exonuclease that degradescapping defective telomeres (see, e.g., Int'l Patent Application No. WO2006/018632).

A senolytic agent described herein may be a polypeptide that is anantibody, or antigen-binding fragment. An antigen-binding fragment maybe an F(ab′)₂, Fab, Fab′, Fv, and Fd and also includes a peptide orpolypeptide that comprises at least one complementary determining region(CDR). The antibody may be an internalizing antibody or antigen-bindingfragment that is internalized by the senescent cell via interaction witha target protein.

Binding properties of an antibody to its cognate antigen, may generallybe determined and assessed using methods that may be readily performedby those having ordinary skill in the art (see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)).As used herein, an antibody is said to be “immunospecific,” “specificfor” or to “specifically bind” to an antigen if it reacts at adetectable level with the polypeptide. Affinities of antibodies andantigen binding fragments thereof can be readily determined usingconventional techniques, for example, those described by Scatchard etal. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)) and by surface plasmonresonance (SPR; BIAcore™, Biosensor, Piscataway, N.J.).

The antibodies may be polyclonal or monoclonal. A variable region or oneor more complementarity determining regions (CDRs) may be identified andisolated from antigen-binding fragment or peptide libraries. Anantibody, or antigen-binding fragment, may be recombinantly engineeredand/or recombinantly produced. An antibody may belong to anyimmunoglobulin class, for example IgG, IgE, IgM, IgD, or IgA and may beobtained from or derived from an animal, for example, fowl (e.g.,chicken) and mammals, which include but are not limited to a mouse, rat,hamster, rabbit, or other rodent, a cow, horse, sheep, goat, camel,human, or other primate. For use in human subjects, antibodies andantigen-binding fragments are typically human, humanized, or chimeric toreduce an immunogenic response by the subject to non-human peptides andpolypeptide sequences.

The antibody may be a monoclonal antibody that is a human antibody,humanized antibody, chimeric antibody, bispecific antibody, or anantigen-binding fragment (e.g., F(ab′)2, Fab, Fab′, Fv, and Fd) preparedor derived therefrom. An antigen-binding fragment may also be anysynthetic or genetically engineered protein (see, e.g., Hayden et al.,Curr Opin. Immunol. 9:201-12 (1997); Coloma et al., Nat. Biotechnol.15:159-63 (1997); U.S. Pat. No. 5,910,573); Holliger et al., CancerImmunol. Immunother. 45:128-30 (1997); Drakeman et al., Expert Opin.Investig. Drugs 6:1169-78 (1997); Koelemij et al., J. Immunother.22:514-24 (1999); Marvin et al., Acta Pharmacol. Sin. 26:649-58 (2005);Das et al., Methods Mol. Med. 109:329-46 (2005); International PatentApplication Nos. PCT/US91/08694 and PCT/US91/04666) and from phagedisplay peptide libraries (see, e.g., Scott et al., Science 249:386(1990); Devlin et al., Science 249:404 (1990); Cwirla et al., Science276: 1696-99 (1997); U.S. Pat. No. 5,223,409; U.S. Pat. No. 5,733,731;U.S. Pat. No. 5,498,530; U.S. Pat. No. 5,432,018; U.S. Pat. No.5,338,665; 1994; U.S. Pat. No. 5,922,545; International ApplicationPublication Nos. WO 96/40987 and WO 98/15833). A peptide that is aminimal recognition unit or a CDR (i.e., any one or more of the threeCDRs present in a heavy chain variable region and/or one or more of thethree CDRs present in a light chain variable region) may be identifiedby computer modeling techniques, which can be used for comparing andpredicting a peptide sequence that will specifically bind to a targetprotein of interest (see, e.g., Bradley et al., Science 309:1868 (2005);Schueler-Furman et al., Science 310:638 (2005)). Useful strategies fordesigning humanized antibodies are described in the art (see, e.g.,Jones et al., Nature 321:522-25 (1986); Riechmann et al., Nature332:323-27 (1988; Padlan et al., FASEB 9:133-39 (1995); Chothia et al.,Nature, 342:377-83 (1989)).

Senescent Cells

The senolytic agents described herein may be used to selectively kill ordestroy a senescent cell in a clinically significant or biologicallysignificant manner. As discussed in detail herein, the one or moresenolytic agents is used in an amount and for a time sufficient thatselectively kills established senescent cells but is insufficient tokill (destroy, cause the death of) a non-senescent cell in a clinicallysignificant or biologically significant manner. The senolytic agents mayselectively kill one or more types of senescent cells (e.g., senescentpreadipocytes, senescent endothelial cells, senescent fibroblasts,senescent neurons, senescent epithelial cells, senescent mesenchymalcells, senescent smooth muscle cells, senescent macrophages, orsenescent chondrocytes).

A senescent cell may exhibit any one or more of the following sevencharacteristics. (1) Senescence growth arrest is essentially permanentand cannot be reversed by known physiological stimuli. (2) Senescentcells increase in size, sometimes enlarging more than twofold relativeto the size of non-senescent counterparts. (3) Senescent cells express asenescence-associated 3-galactosidase (SA-β-gal), which partly reflectsthe increase in lysosomal mass. (4) Most senescent cells expressp161NK4a, which is not commonly expressed by quiescent or terminallydifferentiated cells. (5) Cells that senesce with persistent DDRsignaling harbor persistent nuclear foci, termed DNA segments withchromatin alterations reinforcing senescence (DNA-SCARS). These focicontain activated DDR proteins and are distinguishable from transientdamage foci. DNA-SCARS include dysfunctional telomeres or telomeredysfunction-induced foci (TIF). (6) Senescent cells express and maysecrete molecules associated with senescence, which in certain instancesmay be observed in the presence of persistent DDR signaling, which incertain instances may be dependent on persistent DDR signaling for theirexpression. (7) The nuclei of senescent cells lose structural proteinssuch as Lamin B1 or chromatin-associated proteins such as histones andHMGB1. See, e.g., Freund et al., Mol. Biol. Cell 23:2066-75 (2012);Davalos et al., J. Cell Biol. 201:613-29 (2013); Ivanov et al., J. CellBiol. DOI: 10.1083/jcb.201212110, page 1-15; published online Jul. 1,2013; Funayama et al., J. Cell Biol. 175:869-80 (2006)).

Senescent cells and senescent cell associated molecules can be detectedby techniques and procedures described in the art. For example, thepresence of senescent cells in tissues can be analyzed by histochemistryor immunohistochemistry techniques that detect the senescence marker,SA-beta galactosidase (SA-βgal) (see, e.g., Dimri et al., Proc. Natl.Acad. Sci. USA 92: 9363-9367 (1995)). The presence of the senescentcell-associated polypeptide p16 can be determined by any one of numerousimmunochemistry methods practiced in the art, such as immunoblottinganalysis. Expression of p16 mRNA in a cell can be measured by a varietyof techniques practiced in the art including quantitative PCR. Thepresence and level of senescent cell associated polypeptides (e.g.,polypeptides of the SASP) can be determined by using automated and highthroughput assays, such as an automated Luminex array assay described inthe art (see, e.g., Coppe et al., PLoS Biol 6: 2853-68 (2008)).

The presence of senescent cells can also be determined by detection ofsenescent cell-associated molecules, which include growth factors,proteases, cytokines (e.g., inflammatory cytokines), chemokines,cell-related metabolites, reactive oxygen species (e.g., H₂O₂), andother molecules that stimulate inflammation and/or other biologicaleffects or reactions that may promote or exacerbate the underlyingdisease of the subject. Senescent cell-associated molecules includethose that are described in the art as comprising thesenescence-associated secretory phenotype (SASP, i.e., which includessecreted factors which may make up the pro-inflammatory phenotype of asenescent cell), senescent-messaging secretome, and DNA damage secretoryprogram (DDSP). These groupings of senescent cell associated molecules,as described in the art, contain molecules in common and are notintended to describe three separate distinct groupings of molecules.Senescent cell-associated molecules include certain expressed andsecreted growth factors, proteases, cytokines, and other factors thatmay have potent autocrine and paracrine activities (see, e.g., Coppe etal., supra; Coppe et al. J. Biol. Chem. 281:29568-74 (2006); Coppe etal. PLoS One 5:39188 (2010); Krtolica et al. Proc. Natl. Acad. Sci.U.S.A. 98:12072-77 (2001); Parrinello et al., J. Cell Sci. 118:485-96(2005). ECM associated factors include inflammatory proteins andmediators of ECM remodeling and which are strongly induced in senescentcells (see, e.g., Kuilman et al., Nature Reviews 9:81-94 (2009)). Othersenescent cell-associated molecules include extracellular polypeptides(proteins) described collectively as the DNA damage secretory program(DDSP) (see, e.g., Sun et al., Nature Medicine 18:1359-1368 (2012)).Senescent cell-associated proteins also include cell surface proteins(or receptors) that are expressed on senescent cells, which includeproteins that are present at a detectably lower amount or are notpresent on the cell surface of a non-senescent cell.

Senescence cell-associated molecules include secreted factors which maymake up the pro-inflammatory phenotype of a senescent cell (e.g., SASP).These factors include, without limitation, GM-CSF, GROα, GROα,β,γ,IGFBP-7, IL-1α, IL-6, IL-7, IL-8, MCP-1, MCP-2, MIP-1α, MMP-1, MMP-10,MMP-3, Amphiregulin, ENA-78, Eotaxin-3, GCP-2, GITR, HGF, ICAM-1,IGFBP-2, IGFBP-4, IGFBP-5, IGFBP-6, IL-13, IL-1β, MCP-4, MIF, MIP-3α,MMP-12, MMP-13, MMP-14, NAP2, Oncostatin M, osteoprotegerin, PIGF,RANTES, sgp130, TIMP-2, TRAIL-R3, Acrp30, angiogenin, AxI, bFGF, BLC,BTC, CTACK, EGF-R, Fas, FGF-7, G-CSF, GDNF, HCC-4, I-309, IFN-γ,IGFBP-1, IGFBP-3, IL-1R1, IL-11, IL-15, IL-2R-α, IL-6R, I-TAC, Leptin,LIF, MMP-2, MSP-a, PAI-1, PAI-2, PDGF-BB, SCF, SDF-1, sTNF RI, sTNF RII,Thrombopoietin, TIMP-1, tPA, uPA, uPAR, VEGF, MCP-3, IGF-1, TGF-β3,MIP-1-delta, IL-4, FGF-7, PDGF-BB, IL-16, BMP-4, MDC, MCP-4, IL-10,TIMP-1, Fit-3 Ligand, ICAM-1, AxI, CNTF, INF-γ, EGF, BMP-6. Additionalidentified factors, which include those sometimes referred to in the artas senescence messaging secretome (SMS) factors, some of which areincluded in the listing of SASP polypeptides, include withoutlimitation, IGF1, IGF2, and IGF2R, IGFBP3, IDFBP5, IGFBP7, PAI1, TGF-β,WNT2, IL-1α, IL-6, IL-8, and CXCR2-binding chemokines. Cell-associatedmolecules also include without limitation the factors described in Sunet al., Nature Medicine, supra, and include, including, for example,products of the genes, MMP1, WNT16B, SFRP2, MMP12, SPINK1, MMP10, ENPP5,EREG, BMP6, ANGPTL4, CSGALNACT, CCL26, AREG, ANGPT1, CCK, THBD, CXCL14,NOV, GAL, NPPC, FAM150B, CST1, GDNF, MUCL1, NPTX2, TMEM155, EDN1, PSG9,ADAMTS3, CD24, PPBP, CXCL3, MMP3, CST2, PSG8, PCOLCE2, PSG7, TNFSF15,C17orf67, CALCA, FGF18, IL8, BMP2, MATN3, TFP1, SERPINI 1, TNFRSF25, andIL23A. Senescent cell-associated proteins also include cell surfaceproteins (or receptors) that are expressed on senescent cells, whichinclude proteins that are present at a detectably lower amount or arenot present on the cell surface of a non-senescent cell.

In certain embodiments, senolytic agents that selectively kill at leastsenescent preadipocytes may be useful for treatment of diabetes(particularly type 2 diabetes), metabolic syndrome, or obesity. In otherembodiments, senolytic agents are capable of selectively killing atleast senescent endothelial cells, senescent smooth muscle cells, and/orsenescent macrophages. Such senolytic agents may be useful for treatmentof a cardiovascular disease (e.g., atherosclerosis). In other particularembodiments, the senolytic agents are capable of selectively killing atleast senescent fibroblasts. In still another embodiment, the senolyticagents may selectively kill at least senescent neurons, includingdopamine-producing neurons. In still another embodiment, the senolyticagents may kill at least senescent retinal pigmented epithelial cells orother senescent epithelial cells (e.g., pulmonary senescent epithelialcells or senescent kidney (renal) epithelial cells). Selective killingof at least senescent pulmonary epithelial cells may be useful fortreating pulmonary diseases, such as chronic obstructive pulmonarydisease or idiopathic pulmonary fibrosis. In yet other embodiments, thesenolytic agents may selectively kill at least senescent immune cells(such as senescent macrophages). In still another embodiment, thesenolytic agents may kill at least senescent chondrocytes, which may beuseful for treatment of an inflammatory disorder, such asosteoarthritis.

Methods for Selective Killing of Senescent Cells

Provided herein are methods for selectively killing senescent cells andthereby treating or preventing (reducing the likelihood of occurrenceof) a senescence-associated disease or disorder and comprises use of asenolytic agent as described herein. As described herein, thesesenolytic agents are administered in a manner that would be consideredineffective for treating a cancer. Because the method used for treatinga senescence associated disease with a senolytic agent described hereincomprises one or more of a decreased daily dose, decreased cumulativedose over a single therapeutic cycle, or decreased cumulative dose ofthe senolytic agent (e.g., an MDM2 inhibitor; an inhibitor of at leastone Bcl-2 anti-apoptotic family member that inhibits at least Bcl-xL; anAkt inhibitor) over multiple therapeutic cycles compared with the amountrequired for cancer therapy, the likelihood is decreased that one ormore adverse effects (i.e., side effects) will occur, which adverseeffects are associated with treating a subject according to a regimenoptimized for treating a cancer.

The treatment regimen of the methods for treating a senescenceassociated disease or disorder, comprises administering a senolyticagent for a time sufficient and in an amount sufficient that selectivelykills senescent cells. In certain embodiments, the senolytic agent isadministered within a treatment cycle, which treatment cycle comprises atreatment course followed by a non-treatment interval. A treatmentcourse of administration refers herein to a finite time frame over whichone or more doses of the senolytic agent on one or more days areadministered. The finite time frame may be also called herein atreatment window.

In one embodiment, a method is provided herein for treating asenescence-associated disease or disorder, which is not a cancer, andwhich method comprises administering to a subject in need thereof asmall molecule senolytic agent that selectively kills senescent cellsand is administered within a treatment cycle. In a particularembodiment, the methods comprise administering the senolytic agent in atleast two treatment cycles. In a specific embodiment, the non-treatmentinterval may be at least about 2 weeks or between at least about 0.5-12months, such as at least about one month, at least about 2 months, atleast about 3 months, at least about 4 months, at least about 5 months,at least about 6 months, at least about 7 months, at least about 8months, at least about 9 months, at least about 10 months, at leastabout 11 months, or at least about 12 months (i.e., 1 year). In othercertain particular embodiments, the non-treatment interval is between1-2 years or between 1-3 years, or longer. In certain embodiments, eachtreatment course is no longer than about 1 month, no longer than about 2months, or no longer than about 3 months; or is no longer than 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 26, 27, 28, 29, 30, or 31 days.

In certain embodiments, the treatment window (i.e., treatment course) isonly one day. In other certain embodiments, a single treatment courseoccurs over no longer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 27, 28, 29, 30, or31 days. During such treatment windows, the senolytic agent may beadministered at least on two days (i.e., two days or more) with avariable number of days on which the agent is not administered betweenthe at least two days of administration. Stated another way, within atreatment course when the senolytic agent is administered on two or moredays, the treatment course may have one or more intervals of one or moredays when the senolytic agent, is not administered. By way ofnon-limiting example, when the senolytic agent is administered on 2 ormore days during a treatment course not to exceed 21 days, the agent maybe administered on any total number of days between from 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 26, 27, 28, 29, 30, or 31 days. In certain embodiments, thesenolytic agent is administered to a subject during a treatment courseof 3 days or more, and the agent may be administered every 2^(nd) day(i.e., every other day). In other certain embodiments when the senolyticagent is administered to a subject for a treatment window of 4 days ormore, the senolytic agent may be administered every 3^(rd) day (i.e.,every other third day). In one embodiment, the senolytic agent isadministered on at least two days (i.e., 2 or more) during a treatmentcourse that is at least 2 days and no more than about 21 days (i.e.,from about 2-21 days); at least 2 days and no longer than about 14 days(i.e., from about 2-14 days); at least 2 days and no longer than about10 days (i.e., from about 2-10 days); or at least 2 days and no longerthan about 9 days (i.e., from about 2-9 days); or at least 2 days and nolonger than about 8 days (i.e., from about 2-8 days). In other specificembodiments, the senolytic agent is administered on at least two days(i.e., 2 or more) during a treatment window is at least 2 days and nolonger than about 7 days (i.e., from about 2-7 days); at least 2 daysand no longer than about 6 days (i.e., from about 2-6 days) or at least2 days and no more than about 5 days (i.e., from about 2-5 days) or atleast 2 days and no longer than about 4 days (i.e., from about 2-4days). In yet another embodiment, the treatment window is at least 2days and no longer than 3 days (i.e., 2-3 days), or 2 days. In certainparticular embodiments, the treatment course is no longer than 3 days.In other embodiments, the treatment course is no longer than 5 days. Instill other specific embodiments, the treatment course is no longer than7 days, 10 days, or 14 days or 21 days. In certain embodiments, thesenolytic agent is administered on at least two days (i.e., 2 or moredays) during a treatment window that is at least 2 days and no longerthan about 11 days (i.e., 2-11 days); or the senolytic agent isadministered on at least two days (i.e., 2 or more days) during atreatment window that is at least 2 days and no longer than about 12days (i.e., 2-12 days); or the senolytic agent is administered on atleast two days (i.e., 2 or more days) during a treatment window that isat least 2 days and no more than about 13 days (i.e., 2-13 days); or thesenolytic agent is administered on at least two days (i.e., 2 or moredays) during a treatment course that is at least 2 days and no more thanabout 15 days (i.e., 2-15 days); or the senolytic agent is administeredon at least two days (i.e., 2 or more days) during a treatment coursethat is at least 2 days and no longer than about 16 days, 17 days, 18days, 19 days, or 20 days (i.e., 2-16, 2-17, 2-18, 2-19, 2-20 days,respectively). In other embodiments, the senolytic agent may beadministered on at least 3 days over a treatment course of at least 3days and no longer than any number of days between 3 and 21 days; or isadministered on at least 4 days over a treatment course of at least 4days and no longer than any number of days between 4 and 21 days; or isadministered on at least 5 days over a treatment course of at least 5days and no longer than any number of days between 5 and 21 days; or isadministered on at least 6 days over a treatment course of at least 6days and no longer than any number of days between 6 and 21 days; or isadministered at least 7 days over a treatment course of at least 7 daysand no longer than any number of days between 7 and 21 days; or isadministered at least 8 or 9 days over a treatment course of at least 8or 9 days, respectively, and no longer than any number of days between 8or 9 days, respectively, and 21 days; or is administered at least 10days over a treatment course of at least 10 days and no longer than anynumber of days between 10 and 21 days; or is administered at least 14days over a treatment course of at least 14 days and no longer than anynumber of days between 14 and 21 days; or is administered at least 11 or12 days over a treatment course of at least 11 or 12 days, respectively,and no longer than any number of days between 11 or 12 days,respectively, and 21 days; or is administered at least 15 or 16 daysover a treatment course of at least 15 or 16 days, respectively, and nolonger than any number of days between 15 or 16 days, respectively, and21 days. By way of additional example, when the treatment course is nolonger than 14 days, a senolytic agent may be administered on at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 days over a treatment ofwindow of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 days,respectively, and no longer than 14 days. When the treatment course isno longer than 10 days, a senolytic agent may be administered on atleast 2, 3, 4, 5, 6, 7, 8, 9, or 10 days over a treatment of window ofat least 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, respectively, and no longerthan 10 days. Similarly, when the treatment course is no longer than 7days, a senolytic agent may be administered on at least 2, 3, 4, 5, 6,or 7 days over a treatment window of at least 2, 3, 4, 5, 6, or 7 days,respectively, and no longer than 7 days. In still another example, whenthe treatment course is no longer than 5 days, a senolytic agent may beadministered on at least 2, 3, 4, or 5 days over a treatment of windowof at least 2, 3, 4, or 5 days, respectively, and no longer than 5 days.

With respect to a treatment course of three or more days, doses of thesenolytic agent may be administered for a lesser number of days than thetotal number of days within the particular treatment window. By way ofnon-limiting example, when a course of treatment has a treatment courseof no more than 7, 10, 14, or 21 days, the number of days on which thesenolytic agent may be administered is any number of days between 2 daysand 7, 10, 14, or 21 days, respectively, and at any interval appropriatefor the particular disease being treated, the senolytic agent beingadministered, the health status of the patient and other relevantfactors, which are discussed in greater detail herein. A person skilledin the art will readily appreciate that when the senolytic agent isadministered on two or more days over a treatment window, the agent maybe delivered on the minimum number days of the window, the maximumnumber of days of the window, or on any number of days between theminimum and the maximum.

In certain specific embodiments, a treatment course is one day or thetreatment course is of a length not to exceed 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 days, which are examples of a course wherein thesenolytic agent is administered on two or more days over a treatmentcourse not to exceed (i.e., no longer than) 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 days, respectively. In other certain embodiments, thetreatment course is about 2 weeks (about 14 days or 0.5 months), about 3weeks (about 21 days), about 4 weeks (about one month), about 5 weeks,about 6 weeks (about 1.5 months), about 2 months (or about 60 days), orabout 3 months (or about 90 days). In a particular embodiment, atreatment course is a single daily dosing of the senolytic agent. Inother embodiments, with respect to any treatment course a daily dose ofthe senolytic agent may be as a single administration or the dose may bedivided into 2, 3, 4, or 5 separate administrations to provide the totaldaily dose of the agent.

As described herein, in certain specific embodiments, within a treatmentwindow when the senolytic agent is administered on two are more days,the treatment course may have one or more intervals of one or more dayswhen the senolytic agent, is not administered. Solely as a non-limitingexample, when a treatment window is between two and seven days, a firstdose may be administered on the first day of the treatment window and asecond dose may be administered on the third day of the course, and athird dose may be administered on the seventh day of the treatmentwindow. A person skilled in the art will appreciate that varying dosingschedules may be used during a particular treatment window. In otherspecific embodiments, the senolytic agent is administered daily on eachconsecutive day for the duration of the treatment course. A daily dosemay be administered as a single dose or the daily dose may be dividedinto 2, 3, or 4, or 5 separate administrations to provide the totaldaily dose of the senolytic agent.

In certain embodiments, the treatment course comprises a length of timeduring which the senolytic agent is administered daily. In one specificembodiment, the senolytic agent is administered daily for 2 days. Inanother specific embodiment, the senolytic agent is administered dailyfor 3 days. In yet another particular embodiment, the senolytic agent isadministered daily for 4 days. In one specific embodiment, the senolyticagent is administered daily for 5 days. In yet another particularembodiment, the senolytic agent is administered daily for 6 days. Inanother specific embodiment, the senolytic agent is administered dailyfor 7 days. In yet another particular embodiment, the senolytic agent isadministered daily for 8 days. In still another specific embodiment, thesenolytic agent is administered daily for 9 days. In yet anotherparticular embodiment, the senolytic agent is administered daily for 10days. In yet another particular embodiment, the senolytic agent isadministered daily for 11 days. In yet another particular embodiment,the senolytic agent is administered daily for 12 days. In yet anotherparticular embodiment, the senolytic agent is administered daily for 13days. In yet another particular embodiment, the senolytic agent isadministered daily for 14 days. The treatment window (i.e., course) foreach of the above examples is no longer than 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 days, respectively.

In other specific embodiments, the senolytic agent is administered every2^(nd) day (i.e., every other day) for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, or 14 days. In still other specific embodiments, the senolytic agentis administered every 3^(nd) day (i.e., one day receiving the agentfollowed by two days without receiving the agent) for 4, 5, 6, 7, 8, 9,10, 11, 12, 13, or 14 days. In still other specific embodiments, thesenolytic agent may be administered on every 2^(nd)-3^(rd) day during atreatment window of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. Inyet other embodiments, the senolytic agent may be administered every4^(th) day during a treatment course of 5, 6, 7, 8, 9, 10, 11, 12, 13,or 14 days; or every 5^(th) day during a treatment course of 6, 7, 8, 9,10, 11, 12, 13, or 14 days. A person skilled in the art can readilyappreciate the minimum numbers of days in a treatment window when thesenolytic agent is administered every 6^(th), 7^(th), etc. day over atreatment window of a finite number of days as described herein.

In certain particular embodiments, a senolytic agent may be administereddaily for a longer duration than 14 days and may be administered atleast 15, 16, 17, 18, 19, 20, or at least 21 days. In other specificembodiments, the senolytic agent may be administered daily on each ofthe 15, 16, 17, 18, 19, 20, or 21 days. In another specific embodiment,the senolytic agent may be administered every second day during atreatment window of 15, 16, 17, 18, 19, 20, or 21 days. In anotherspecific embodiment, the senolytic agent may be administered every thirdday during a treatment window of 15, 16, 17, 18, 19, 20, or 21 days. Instill other specific embodiments, the senolytic agent may beadministered on every 2^(nd)-3^(rd) day during a treatment window of 15,16, 17, 18, 19, 20, or 21 days. In yet other embodiments, the senolyticagent may be administered every 4^(th) day during a treatment course of15, 16, 17, 18, 19, 20, or 21 days; or every 5^(th) day during atreatment course of 15, 16, 17, 18, 19, 20, or 21 days. A person skilledin the art can readily appreciate the minimum numbers of days in atreatment window when the senolytic agent is administered every 6^(th),7^(th), etc. day over a treatment window of a finite number of days asdescribed herein.

In another certain particular embodiment, a senolytic agent may beadministered daily for a longer duration than 14 days and may beadministered at least 15, 16, 17, 18, 19, 20, or at least 21 days. Inother specific embodiments, the senolytic agent may be administereddaily on each of the 15, 16, 17, 18, 19, 20, or 21 days. In anotherspecific embodiment, the senolytic agent may be administered everysecond day during a treatment window of 15, 16, 17, 18, 19, 20, or 21days. In another specific embodiment, the senolytic agent may beadministered every third day during a treatment window of 15, 16, 17,18, 19, 20, or 21 days. In still other specific embodiments, thesenolytic agent may be administered on every 2^(nd)-3^(rd) day during atreatment window of 15, 16, 17, 18, 19, 20, or 21 days. In yet otherembodiments, the senolytic agent may be administered every 4^(th) dayduring a treatment course of 15, 16, 17, 18, 19, 20, or 21 days; orevery 5^(th) day during a treatment course of 15, 16, 17, 18, 19, 20, or21 days. A person skilled in the art can readily appreciate the minimumnumbers of days in a treatment window when the senolytic agent isadministered every 6^(th), 7^(th), etc. day over a treatment window of afinite number of days as described herein.

In another certain particular embodiment, a senolytic agent may beadministered in a treatment course daily for a longer duration than 14days or 21 days and may be administered in a treatment course of aboutone month, about two months, or about three months. In other specificembodiments, the senolytic agent may be administered daily on each of aone month, two month, or three month treatment course. In anotherspecific embodiment, the senolytic agent may be administered everysecond day during a treatment course of about one month, about twomonths, or about three months. In another specific embodiment, thesenolytic agent may be administered every third day during a treatmentcourse of about one month, about two months, or about three months. Instill other specific embodiments, the senolytic agent may beadministered on every 2^(nd)-3^(rd) day during a treatment course ofabout one month, about two months, or about three months. In yet otherembodiments, the senolytic agent may be administered every 4^(th) dayduring a treatment course of about one month, about two months, or aboutthree months; or every 5^(th) day during a treatment course of about onemonth, about two months, or about three months s. A person skilled inthe art can readily appreciate the minimum numbers of days in atreatment course when the senolytic agent is administered every 6^(th),7^(th), etc. day over a treatment window of a finite number of days asdescribed herein.

By way of non-limiting example, a longer treatment window with adecreased dose per day may be a treatment option for a subject. In otherparticular embodiments and by way of example, the stage or severity ofthe senescence associated disease or disorder or other clinical factormay indicate that a longer term course may provide clinical benefit. Incertain embodiments, the senolytic agent is administered daily, oroptionally, every other day (every 2^(nd) day) or every 3^(rd) day, orgreater interval (i.e., every 4^(th) day, 5^(th) day, 6^(th) day) duringa treatment course of about 1-2 weeks (e.g., about 5-14 days), about 1-3weeks (e.g., about 5-21 days), about 1-4 weeks (e.g., about 5-28 days,about 5-36 days, or about 5-42 days, 7-14 days, 7-21 days, 7-28 days,7-36 days, or 7-42 days; or 9-14 days, 9-21 days, 9-28 days, 9-36 days,or 9-42 days. In other certain embodiments, the treatment course isbetween about 1-3 months. In a specific embodiment, the senolytic agentis administered daily for at least five days, and in another particularembodiment, the senolytic agent is administered daily for 5-14 days. Inother particular embodiments, the senolytic agent is administered for atleast seven days, for example, for 7-14, 7-21, 7-28 days, 7-36 days, or7-42 days. In other particular embodiments, the senolytic agent isadministered for at least nine days, for example, for 9-14 days, 9-21days, 9-28 days, 9-36 days, or 9-42 days.

Even though as discussed herein and above, a treatment course comprisingadministering a senolytic agent provides clinical benefit, in othercertain embodiments, a treatment course is repeated with a time intervalbetween each treatment course when the senolytic agent is notadministered (i.e., non-treatment interval, off-drug treatment). Atreatment cycle as described herein and in the art comprises a treatmentcourse followed by a non-treatment interval. A treatment cycle may berepeated as often as needed. For example, a treatment cycle may berepeated at least once, at least twice, at least three times, at leastfour times, at least five times, or more often as needed. In certainspecific embodiments, a treatment cycle is repeated once (i.e.,administration of the senolytic agent comprises 2 treatment cycles). Inother certain embodiments, the treatment cycle is repeated twice orrepeated 3 or more times. Accordingly, in certain embodiments, one, two,three, four, five, six, seven, eight, nine, ten, or more treatmentcycles of treatment with a senolytic agent are performed. In particularembodiments, a treatment course or a treatment cycle may be repeated,such as when the senescence associated disease or disorder recurs, orwhen symptoms or sequelae of the disease or disorder that weresignificantly diminished by one treatment course as described above haveincreased or are detectable, or when the symptoms or sequelae of thedisease or disorder are exacerbated, a treatment course may be repeated.In other embodiments when the senolytic agent is administered to asubject to prevent (i.e., reduce likelihood of occurrence ordevelopment) or to delay onset, progression, or severity of senescenceassociated disease or disorder, a subject may receive the senolyticagent over two or more treatment cycles. Accordingly, in certainembodiments, one cycle of treatment is followed by a subsequent cycle oftreatment. Each treatment course of a treatment cycle or each treatmentcourse of two or more treatment cycles are typically the same induration and dosing of the senolytic agent. In other embodiments, theduration and dosing of the senolytic agent during each treatment courseof a treatment cycle may be adjusted as determined by a person skilledin the medical art depending, for example, on the particular disease ordisorder being treated, the senolytic agent being administered, thehealth status of the patient and other relevant factors, which arediscussed in greater detail herein. Accordingly, a treatment course of asecond or any subsequent treatment cycle may be shortened or lengthenedas deemed medically necessary or prudent. In other words, as would beappreciated by a person skilled in the art, each treatment course of twoor more treatment cycles are independent and the same or different; andeach non-treatment interval of each treatment cycle is independent andthe same or different.

As described herein, each course of treatment in a treatment cycle isseparated by a time interval of days, weeks, or months without treatmentwith a senolytic agent (i.e., non-treatment time interval or off-druginterval; called non-treatment interval herein). The non-treatmentinterval (such as days, weeks, months) between one treatment course anda subsequent treatment course is typically greater than the longest timeinterval (i.e., number of days) between any two days of administrationin the treatment course. By way of example, if a treatment course is nolonger than 14 days and the agent is administered every other day duringthis treatment course, the non-treatment interval between two treatmentcourses is greater than 2 days, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, or 14 days or about 3 weeks, about 4 weeks, about 6 weeks, or about2 months or longer as described herein. In particular embodiments, thenon-treatment interval between two treatment courses is about 5 days,about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 6weeks, about 2 months (8 weeks), about 3 months, about 4 months, about 5months, about 6 months, about 7 months, about 8 months, about 9 months,about 10 months, about 11 months, about 12 months (about 1 year), about18 months (about 1.5 years), or longer. In certain specific embodiments,the non-treatment interval is about 2 years or about 3 years. In certainspecific embodiments, the non-treatment time interval is at least about14 days, at least about 21 days, at least about 1 month, at least about2 months, at least about 3 months, at least about 4 months, at leastabout 5 months, at least about 6 months, or at least about 1 year. Incertain embodiments, a course of treatment (whether daily, every otherday, every 3^(rd) day, or other interval between administrations withinthe treatment course as described above (e.g., 1-14 days, 2-14 days,2-21 days, or 1-21 days)) is administered about every 14 days (i.e.,about every 2 weeks) (i.e., 14 days without senolytic agent treatment),about every 21 days (i.e., about every 3 weeks), about every 28 days(i.e., about every 4 weeks), about every one month, about every 36 days,about every 42 days, about every 54 days, about every 60 days, or aboutevery month (about every 30 days), about every two months (about every60 days), about every quarter (about every 90 days), or aboutsemi-annually (about every 180 days). In other certain embodiments, acourse of treatments (e.g., byway of non-limiting example,administration on at least one day or on at least two days during acourse for about 2-21 days, about 2-14, days, about 5-14 days, about7-14 days, about 9-14 days, about 5-21 days, about 7-21 days, about 9-21days) is administered every 28 days, every 36 days, every 42 days, every54 days, every 60 days, or every month (about every 30 days), every twomonths (about every 60 days), every quarter (about every 90 days), orsemi-annually (about every 180 days), or about every year (about 12months). In other embodiments, a course of treatment (such as by way ofnon-limiting examples, e.g., for about 5-28 days, about 7-28 days, orabout 9-28 days whether daily, every other day, every 3^(rd) day, orother interval between administrations within the treatment course) isadministered every 36 days, 42 days, 54 days, 60 days, or every month(about every 30 days), every two months (about every 60 days), everyquarter (about every 90 days), or semi-annually (about every 180 days).In other particular embodiments, a course of treatment (e.g., for about5-36 days, 7-36 days, or 9-36 days whether daily, every other day, every3^(rd) day, or other interval between administrations within thetreatment course) is administered every 42 days, 54 days, 60 days, orevery month (about every 30 days), every two months (about every 60days), every quarter (about every 90 days), or semi-annually (aboutevery 180 days), or about every year (about 12 months).

In a particular embodiment, the treatment course is one day and thenon-treatment interval is at least about 14 days, about 21 days, about 1month, about 2 months (8 weeks), about 3 months, about 4 months, about 5months, about 6 months, about 7 months, about 8 months, about 9 months,about 10 months, about 11 months, about 12 months (about 1 year), about18 months (about 1.5 years), or longer. In other certain embodiments,the treatment course is at least two days or is at least 3 days and nolonger than 10 days, and the non-treatment interval is at least about 14days, about 21 days, about 1 month, about 2 months (8 weeks), about 3months, about 4 months, about 5 months, about 6 months, about 7 months,about 8 months, about 9 months, about 10 months, about 11 months, about12 months (about 1 year), about 18 months (about 1.5 years), or longer.In still another embodiment, the treatment course is at least three daysand no longer than 10 days, no longer than 14 days, or no longer than 21days, and the non-treatment interval is at least about 14 days, about 21days, about 1 month, about 2 months (8 weeks), about 3 months, about 4months, about 5 months, about 6 months, about 7 months, about 8 months,about 9 months, about 10 months, about 11 months, about 12 months (about1 year), about 18 months (about 1.5 years), or longer. In still anotherembodiment, a treatment course (e.g., for about 5-42, 7-42, or 9-42 dayswhether daily, every other day, every 3^(rd) day, or other intervalbetween administrations within the treatment course) is administeredevery 42 days, 60 days, or every month (about every 30 days), every twomonths (about every 60 days), every quarter (about every 90 days), orsemi-annually (about every 180 days), or about every year (about 12months). In a particular embodiment, the senolytic agent is administereddaily for 5-14 days every 14 days (about every 2 weeks), or every 21-42days. In another particular embodiment, the senolytic agent isadministered daily for 5-14 days quarterly. In another particularembodiment, the senolytic agent is administered daily for 7-14 daysevery 21-42 days. In another particular embodiment, the senolytic agentis administered daily for 7-14 days quarterly. In still other particularembodiments, the senolytic agent is administered daily for 9-14 daysevery 21-42 days or every 9-14 days quarterly. In still otherembodiments, the non-treatment interval may vary between treatmentcourses. By way of non-limiting example, the non-treatment interval maybe 14 days after the first course of treatment and may be 21 days orlonger after the second, third, or fourth (or more) course of treatment.In other particular embodiments, the senolytic agent is administered tothe subject in need thereof once every 0.5-12 months. In other certainembodiments, the senolytic agent is administered to the subject in needonce every 4-12 months.

In certain embodiments, a senolytic agent is administered to a subjectto reduce the likelihood or the risk that the subject will develop aparticular disorder or to delay onset of one or more symptoms of asenescence-associated disease or disorder. In certain embodiments, thesenolytic agent is administered for one or more days (e.g., any numberof consecutives days between and including 2-3, -4, -5, -6, -7, -8, -9,-10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, and 2-21 days)every 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In a particularembodiment, the senolytic agent is administered for one or more days(e.g., any number of consecutives days between and including 1-9 days)every 5 or 6 months.

Without wishing to be bound by any particular theory, periodicadministration of the senolytic agent kills newly formed senescent cellsand thereby reduces (decreases, diminishes) the total number ofsenescent cells accumulating in the subject. In another embodiment, thetotal number of senescent cells accumulating in the subject is decreasedor inhibited by administering the senolytic agent once or twice weeklyor according to any of the other treatment courses described above. Thetotal daily dose of a senolytic agent may be delivered as a single doseor as multiple doses on each day of administration. In other certainparticular embodiments, when multiple cycles of the senolytic agent areadministered, the dose of a senolytic agent administered on a single daymay be less than the daily dose administered if only a single treatmentcourse is intended to be administered.

In certain embodiments, method for treating a senescence-associateddisease or disorder comprising administering to a subject in needthereof a small molecule senolytic agent that selectively killssenescent cells; wherein the senescence-associated disease or disorderis not a cancer, and wherein the senolytic agent is administered withinone or two treatment cycles, typically two treatment cycles. In certainspecific embodiments, the non-treatment interval is at least 2 weeks andeach treatment course is no longer than 3 months.

Also provided herein are methods for selectively killing a senescentcell comprising contacting the senescent cell with a senolytic agentdescribed herein (i.e., facilitating interaction or in some mannerallowing the senescent cell and senolytic agent to interact) underconditions and for a time sufficient to kill the senescent cell. In suchembodiments, the agent selectively kills senescent cells overnon-senescent cells (i.e., the agent selectively kills senescent cellscompared with killing of non-senescent cells). In certain embodiments,the senescent cell to be killed is present in a subject (e.g., a humanor non-human animal). The senolytic agent(s) may be administered to thesubject according to the treatment cycles, treatment courses, andnon-treatment intervals described above and herein.

In particular embodiments, a single (i.e., only, sole) senolytic agentis administered to the subject for treating a senescence-associateddisease or disorder. In certain embodiments, administration of a singlesenolytic agent may be sufficient and clinically beneficial to treat asenescence-associated disease or disorder. Accordingly, in certainparticular embodiments, a senolytic agent is administered as amonotherapy and is the single (i.e., only, sole) active agentadministered to the subject for treating the condition or disease.Medications that are not necessarily excluded from administration to thesubject when a senolytic agent is administered as a monotherapy include,by way of non-limiting examples, medications for other purposes such aspalliative care or comfort (e.g., aspirin, acetominophen, ibuprofen, orprescription pain-killers; anti-itching topical medications) or fortreating a different disease or condition, especially if the othermedications are not senolytic agents, such as drugs for loweringcholesterol, statins, eye wetting agents, and other such medicationsfamiliar to a person skilled in the medical art.

In specific embodiments, if the senolytic agent is an MDM2 inhibitor,the MDM2 inhibitor is administered as a monotherapy (i.e., the onlyactive therapeutic agent), and each treatment course is at least 5 dayslong during which course the MDM2 inhibitor is administered on at least5 days. In certain other embodiments, the MDM2 inhibitor is administeredon at least 9 days. In still more specific embodiments, the MDM2inhibitor is Nutlin-3a.

The dosing regimens, treatment courses, and treatment cycles (can bereviewed and modified or adjusted, continued or discontinued, asdetermined by a person skilled in the art, depending on theresponsiveness of the subject to the senolytic agent, the stage of thedisease, the general health of the subject, and other factors that aredescribed herein and in the art.

As described herein, certain senolytic agents that may be used in themethods have been described as useful or potentially useful for treatinga cancer; however, in embodiments of the methods for treating asenescence associated disorder or disease, the senolytic agents areadministered in a manner that would be considered different and likelyineffective for treating a cancer. Accordingly, the methods describedherein are useful for treating a senescence-associated disorder ordisease but are not described as also useful as a primary therapy (aloneor with another chemotherapy agent or radiotherapy) for treating acancer. In one embodiment, the method used for treating a senescenceassociated disease or disorder with a senolytic agent may comprise adecreased daily dose compared with the daily dose of the agent asrequired for cancer therapy. In another embodiment, the method used fortreating a senescence associated disease or disorder with a senolyticagent described herein may comprise decreased cumulative dose over asingle treatment cycle compared with the cumulative dose of the agent asrequired for cancer therapy. In still another embodiment, the methodused for treating a senescence associated disease or disorder with asenolytic agent described herein may comprise or decreased cumulativedose of the agent administered over multiple treatment cycles comparedwith the dose of the agent as required for multiple cancer therapycycles.

By way of example, in certain embodiments, when the senolytic agent isan agent that can be cytotoxic to cancer cells and may be used in theoncology art in a manner for treating a cancer (for example, an MDM2inhibitor (e.g., Nutlin-3a; RG-7112) or an inhibitor of one or moreBCL-2 anti-apoptotic protein family members and which inhibits at leastBcl-xL (e.g., ABT-263, ABT-737, WEHI-539, A-1155463)), the methods fortreating a senescence associated disease or disorder compriseadministering the senolytic agent in one or two or more treatmentcycles, and the total dose of the senolytic agent administered duringeach treatment course, each treatment cycle, and/or cumulatively overtwo or more treatment cycles is an amount less than the amount effectivefor a cancer treatment. The amount of such a senolytic agentadministered to a subject over a given time period (such as one week,two weeks, one month, six months, one year) for treating a senescenceassociated disease or disorder, for example, may be about from a 20-folddecrease to about a 5000-fold decrease in total amount compared with thetotal amount of the same agent administered to a subject who isreceiving the agent for treatment of a cancer. The fold decrease in theamount (i.e., lesser amount) of the senolytic agent administered over agiven time period (i.e., number of days, months, years) for treating asenescence associated disease or disorder may be about a 20-folddecrease, about a 25-fold decrease, about a 30-fold decrease, about a40-fold decrease, about a 50-fold decrease, about a 60-fold decrease,about a 75-fold decrease, about a 100-fold decrease, about a 125-folddecrease, about a 150-fold decrease, about a 175-fold decrease, about a200-fold decrease, about a 300-fold decrease, about a 400-fold decrease,about a 500-fold decrease, about a 750-fold decrease, about a 1000-folddecrease, about a 1250-fold decrease, about a 1500-fold decrease, abouta 1750-fold decrease, about a 2000-fold decrease, about a 2250-folddecrease, about a 2500-fold decrease, about a 2750-fold decrease, abouta 3000-fold decrease, about a 3250-fold decrease, about a 3500-folddecrease, about a 3750-fold decrease, about a 3000-fold decrease, abouta 3500-fold decrease, about a 4000-fold decrease, about a 4500-folddecrease, or about a 5000-fold decrease compared with the amount of theagent administered to a subject for treating a cancer over the samelength of time. A lower dose required for treating a senescenceassociated disease may also be attributable to the route ofadministration. For example, when a senolytic agent is used for treatinga senescence-associated pulmonary disease or disorder (e.g., COPD, IPF),the senolytic agent may be delivered directly to the lungs (e.g., byinhalation, by intubation, intranasally, or intratracheally), and alower dose per day and/or per treatment course is required than if theagent were administered orally. Also, by way of another example, when asenolytic agent is used for treating osteoarthritis or asenescence-associated dermatological disease or disorder, the senolyticagent may be delivered directly to the osteoarthritic joint (e.g.,intra-articularly, intradermally, topically, transdermally) or to theskin (e.g., topically, subcutaneously, intradermally, transdermally),respectively, at a lower does per day and/or per treatment course thanif the senolytic agent were administered orally. When a senolytic agentis delivered orally, for example, the dose of the senolytic agent perday may be the same amount as administered to a patient for treating acancer; however, the amount of the agent that is delivered over atreatment course or treatment cycle is significantly less than theamount administered to a subject who receives the appropriate amount ofthe agent for treating a cancer.

In certain embodiments, the methods described herein comprise using thesenolytic agent in an amount that is a reduced amount compared with theamount that may be delivered systemically, for example, orally orintravenously to a subject who receives the senolytic agent when theagent is used for treating a cancer. In certain specific embodiments,methods of treating a senescence-associated disease or disorder byselectively killing senescent cells comprises administering thesenolytic agent at a dose that is at least 10% (i.e., one-tenth), atleast 20% (one-fifth), 25% (one-fourth), 30%-33% (about one-third), 40%(two-fifths), or at least 50% (half) of the dose that is administered toa subject who has cancer for killing cancer cells during a treatmentcourse, a treatment cycle, or two or more treatment cycles that form thecancer therapy protocol (i.e., regimen). In other particularembodiments, the dose of the senolytic agent(s) used in the methodsdescribed herein is at least 60%, 70%, 80%, 85%, 90%, or 95% of the dosethat is administered to a subject who has cancer. The therapeuticregimen, comprising the dose of senolytic agent and schedule and mannerof administration that may be used for treating a senescence-associateddisorder or disease is also a regimen insufficient to be significantlycytotoxic to non-senescent cells.

In certain embodiments, a method for treating a senescence-associateddisease or disorder that is not a cancer comprises administering to asubject in need thereof a therapeutically effective amount of a smallmolecule senolytic agent that selectively kills senescent cells (i.e.,selectively kills senescent cells over non-senescent cells or comparedwith non-senescent cells) and which agent is cytotoxic to cancer cells,wherein the senolytic agent is administered within at least onetreatment cycle, which treatment cycle comprises a treatment coursefollowed by a non-treatment interval. The total dose of the senolyticagent administered during the treatment course, and/or the total dose ofthe senolytic agent administered during the treatment cycle, and/or thetotal dose of the senolytic agent administered during two or moretreatment cycles is an amount less than the amount effective for acancer treatment. In certain embodiments, the senolytic agent is aninhibitor of a Bcl-2 anti-apoptotic protein family member that inhibitsat least Bcl-xL; an MDM2 inhibitor; or an Akt specific inhibitor.Examples of these inhibitors are described herein. In other certainembodiments, the senolytic agent is administered as a monotherapy, andis the single active senolytic agent administered to the subject fortreating the disease or disorder. The number of days in the treatmentcourse and the treatment interval are described in detail herein.

In one embodiment, a method is provided herein for treating asenescence-associated disease or disorder, wherein thesenescence-associated disease is not cancer and the method comprisesadministering to a subject in need thereof a senolytic agent or smallmolecule senolytic compound that selectively kills senescent cells, andthe administration is for a short duration (e.g., shorter than may beused for a particular agent for treating a cancer), such as a singleday, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, or 15 days. In theseparticular embodiments, this treatment course on any number of daysbetween 1-15 days is a single treatment course and is not repeated. Inanother particular embodiment, a senolytic agent is administered for 16days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 daysas a single treatment course that is not repeated.

In certain specific embodiments, the senolytic agent is ABT-263(navitoclax). In some embodiments, navitoclax is administered in atreatment window comprising 21 days. In some embodiments, navitoclax isadministered daily for 14 days followed by 7 days off. In someembodiments, navitoclax is administered daily for 13 days followed by 8days off. In some embodiments, navitoclax is administered daily for 12days followed by 9 days off. In some embodiments, navitoclax isadministered daily for 11 days followed by 10 days off. In someembodiments, navitoclax is administered daily for 10 days followed by 11days off. In some embodiments, navitoclax is administered daily for 9days followed by 12 days off. In some embodiments, navitoclax isadministered daily for 8 days followed by 13 days off. In someembodiments, navitoclax is administered daily for 7 days followed by 14days off. In some embodiments, navitoclax is administered daily for 6days followed by 15 days off. In some embodiments, navitoclax isadministered daily for 5 days followed by 16 days off. In someembodiments, navitoclax is administered daily for 4 days followed by 17days off. In some embodiments, navitoclax is administered daily for 3days followed by 18 days off. In some embodiments, navitoclax isadministered daily for 2 days followed by 19 days off. In someembodiments, navitoclax is administered for 1 day followed by 20 daysoff.

In some embodiments, navitoclax is administered daily for 21 days in adose of about 150 mg to 325 mg. In some embodiments, navitoclax isadministered daily for 21 days in a dose of about 150 mg to 300 mg. Insome embodiments, navitoclax is administered daily for 21 days in a doseof about 150 mg to 275 mg. In some embodiments, navitoclax isadministered daily for 21 days in a dose of about 150 mg to 250 mg. Insome embodiments, navitoclax is administered daily for 21 days in a doseof about 150 mg to 225 mg. In some embodiments, navitoclax isadministered daily for 21 days in a dose of about 150 mg to 200 mg. Insome embodiments, navitoclax is administered daily for 21 days in a doseof about 150 mg to 175 mg. In some embodiments, navitoclax isadministered daily for 21 days in a dose of about 150 mg. In someembodiments, navitoclax is administered daily for 21 days in a dose ofabout 125 mg. In some embodiments, navitoclax is administered daily for21 days in a dose of about 100 mg. In some embodiments, navitoclax isadministered daily for 21 days in a dose of about 75 mg. In someembodiments, navitoclax is administered daily for 21 days in a dose ofabout 50 mg. In some embodiments, navitoclax is administered daily for21 days in a dose of about 25 mg.

In some embodiments, navitoclax is administered daily for 14 days in adose of about 150 mg to 325 mg. In some embodiments, navitoclax isadministered daily for 14 days in a dose of about 150 mg to 300 mg. Insome embodiments, navitoclax is administered daily for 14 days in a doseof about 150 mg to 275 mg. In some embodiments, navitoclax isadministered daily for 14 days in a dose of about 150 mg to 250 mg. Insome embodiments, navitoclax is administered daily for 14 days in a doseof about 150 mg to 225 mg. In some embodiments, navitoclax isadministered daily for 14 days in a dose of about 150 mg to 200 mg. Insome embodiments, navitoclax is administered daily for 14 days in a doseof about 150 mg to 175 mg. In some embodiments, navitoclax isadministered daily for 14 days in a dose of about 150 mg. In someembodiments, navitoclax is administered daily for 14 days in a dose ofabout 125 mg. In some embodiments, navitoclax is administered daily for14 days in a dose of about 100 mg. In some embodiments, navitoclax isadministered daily for 14 days in a dose of about 75 mg. In someembodiments, navitoclax is administered daily for 14 days in a dose ofabout 50 mg. In some embodiments, navitoclax is administered daily for14 days in a dose of about 25 mg.

In some embodiments, navitoclax is administered daily for 7 days in adose of about 150 mg to 325 mg. In some embodiments, navitoclax isadministered daily for 7 days in a dose of about 150 mg to 300 mg. Insome embodiments, navitoclax is administered daily for 7 days in a doseof about 150 mg to 275 mg. In some embodiments, navitoclax isadministered daily for 7 days in a dose of about 150 mg to 250 mg. Insome embodiments, navitoclax is administered daily for 7 days in a doseof about 150 mg to 225 mg. In some embodiments, navitoclax isadministered daily for 7 days in a dose of about 150 mg to 200 mg. Insome embodiments, navitoclax is administered daily for 7 days in a doseof about 150 mg to 175 mg. In some embodiments, navitoclax isadministered daily for 7 days in a dose of about 150 mg. In someembodiments, navitoclax is administered daily for 7 days in a dose ofabout 125 mg. In some embodiments, navitoclax is administered daily for7 days in a dose of about 100 mg. In some embodiments, navitoclax isadministered daily for 7 days in a dose of about 75 mg. In someembodiments, navitoclax is administered daily for 7 days in a dose ofabout 50 mg. In some embodiments, navitoclax is administered daily for 7days in a dose of about 25 mg. In other particular embodiments, theabove doses are administered daily for 1, 2, 3, 4, 5, or 6 days, 8, 9,10, 11, 12, 13, 15, 16, 17, 18, 19, or 20 days.

In some embodiments, the senolytic agent is nutlin-3a. In someembodiments, nutlin-3a is administered in a treatment window comprising28 days. In some embodiments, nutlin-3a is administered daily for 10days, followed by 18 days off. In some embodiments, nutlin-3a isadministered daily for 9 days, followed by 19 days off. In someembodiments, nutlin-3a is administered daily for 8 days, followed by 20days off. In some embodiments, nutlin-3a is administered daily for 7days, followed by 21 days off. In some embodiments, nutlin-3a isadministered daily for 6 days, followed by 22 days off. In someembodiments, nutlin-3a is administered daily for 5 days, followed by 23days off. In some embodiments, nutlin-3a is administered daily for 4days, followed by 24 days off. In some embodiments, nutlin-3a isadministered daily for 3 days, followed by 25 days off. In someembodiments, nutlin-3a is administered daily for 2 days, followed by 26days off. In some embodiments, nutlin-3a is administered for 1 day,followed by 27 days off.

In some specific embodiments, nutlin-3a is administered daily for 10days in a dose of about 20 mg/m². In some embodiments, nutlin-3a isadministered daily for 10 days in a dose of about 19 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 18 mg/m². In some embodiments, nutlin-3a is administered daily for10 days in a dose of about 17 mg/m². In some embodiments, nutlin-3a isadministered daily for 10 days in a dose of about 16 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 15 mg/m². In some embodiments, nutlin-3a is administered daily for10 days in a dose of about 14 mg/m². In some embodiments, nutlin-3a isadministered daily for 10 days in a dose of about 13 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 12 mg/m². In some embodiments, nutlin-3a is administered daily for10 days in a dose of about 11 mg/m². In some embodiments, nutlin-3a isadministered daily for 10 days in a dose of about 10 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 9 mg/m². In some embodiments, nutlin-3a is administered daily for10 days in a dose of about 8 mg/m². In some embodiments, nutlin-3a isadministered daily for 10 days in a dose of about 7 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 6 mg/m². In some embodiments, nutlin-3a is administered daily for10 days in a dose of about 5 mg/m². In some embodiments, nutlin-3a isadministered daily for 10 days in a dose of about 4 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 3 mg/m². In some embodiments, nutlin-3a is administered daily for10 days in a dose of about 2 mg/m². In some embodiments, nutlin-3a isadministered daily for 10 days in a dose of about 1 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 0.75 mg/m². In some embodiments, nutlin-3a is administered dailyfor 10 days in a dose of about 0.5 mg/m². In some embodiments, nutlin-3ais administered daily for 10 days in a dose of about 0.25 mg/m². In someembodiments, nutlin-3a is administered daily for 10 days in a dose ofabout 0.1 mg/m². In some embodiments, nutlin-3a is administered dailyfor 10 days in a dose of about 0.01 mg/m². In certain embodiments,nutlin-3a is administered for 5, 6, 7, 8, 9, 11, 12, 13, or for 14 daysat the doses described above.

Senescence-Associated Diseases and Disorders

Methods are provided herein for treating conditions, diseases, ordisorders related to, associated with, or caused by cellular senescence,including age-related diseases and disorders in a subject in needthereof. A senescence-associated disease or disorder may also be calledherein a senescent cell-associated disease or disorder.Senescence-associated diseases and disorders include, for example,cardiovascular diseases and disorders, inflammatory diseases anddisorders, autoimmune diseases and disorders, pulmonary diseases anddisorders, eye diseases and disorders, metabolic diseases and disorders,neurological diseases and disorders (e.g., neurodegenerative diseasesand disorders); age-related diseases and disorders induced bysenescence; skin conditions; age-related diseases; dermatologicaldiseases and disorders; and transplant related diseases and disorders. Aprominent feature of aging is a gradual loss of function, ordegeneration that occurs at the molecular, cellular, tissue, andorganismal levels. Age-related degeneration gives rise towell-recognized pathologies, such as sarcopenia, atherosclerosis andheart failure, osteoporosis, pulmonary insufficiency, renal failure,neurodegeneration (including macular degeneration, Alzheimer's disease,and Parkinson's disease), and many others. Although different mammalianspecies vary in their susceptibilities to specific age-relatedpathologies, collectively, age-related pathologies generally rise withapproximately exponential kinetics beginning at about the mid-point ofthe species-specific life span (e.g., 50-60 years of age for humans)(see, e.g., Campisi, Annu. Rev. Physiol. 75:685-705 (2013); Naylor etal., Clin. Pharmacol. Ther. 93:105-16 (2013)).

Examples of senescence-associated conditions, disorders, or diseasesthat may be treated by administering any one of the senolytic agentsdescribed herein according to the methods described herein include,cognitive diseases (e.g., mild cognitive impairment (MCI), Alzheimer'sdisease and other dementias; Huntington's disease); cardiovasculardisease (e.g., atherosclerosis, cardiac diastolic dysfunction, aorticaneurysm, angina, arrhythmia, cardiomyopathy, congestive heart failure,coronary artery disease, myocardial infarction, endocarditis,hypertension, carotid artery disease, peripheral vascular diseases,cardiac stress resistance, cardiac fibrosis); metabolic diseases anddisorders (e.g., obesity, diabetes, metabolic syndrome); motor functiondiseases and disorders (e.g., Parkinson's disease, motor neurondysfunction (MND); Huntington's disease); cerebrovascular disease;emphysema; osteoarthritis; benign prostatic hypertrophy; pulmonarydiseases (e.g., idiopathic pulmonary fibrosis, chronic obstructivepulmonary disease (COPD), emphysema, obstructive bronchiolitis, asthma);inflammatory/autoimmune diseases and disorders (e.g., osteoarthritis,eczema, psoriasis, osteoporosis, mucositis, transplantation relateddiseases and disorders); ophthalmic diseases or disorders (e.g.,age-related macular degeneration, cataracts, glaucoma, vision loss,presbyopia); diabetic ulcer; metastasis; a chemotherapeutic side effect,a radiotherapy side effect; aging-related diseases and disorders (e.g.,kyphosis, renal dysfunction, frailty, hair loss, hearing loss, musclefatigue, skin conditions, sarcopenia, and herniated intervertebral disc)and other age-related diseases that are induced by senescence (e.g.,diseases/disorders resulting from irradiation, chemotherapy, smokingtobacco, eating a high fat/high sugar diet, and environmental factors);wound healing; skin nevi; fibrotic diseases and disorders (e.g., cysticfibrosis, renal fibrosis, liver fibrosis, pulmonary fibrosis, oralsubmucous fibrosis, cardiac fibrosis, and pancreatic fibrosis). Incertain embodiments, any one or more of the diseases or disordersdescribed above or herein may be excluded.

In a more specific embodiment, methods are provided for treating asenescence-associated disease or disorder by killing senescent cells(i.e., established senescent cells) associated with the disease ordisorder in a subject who has the disease or disorder by administering asenolytic agent, wherein the disease or disorder is osteoarthritis;idiopathic pulmonary fibrosis; chronic obstructive pulmonary disease(COPD); or atherosclerosis.

Subjects (i.e., patients, individuals (human or non-human animals)) whomay benefit from use of the methods described herein that compriseadministering a senolytic agent include those who may also have acancer. The subject treated by these methods may be considered to be inpartial or complete remission (also called cancer remission). Asdiscussed in detail herein, the senolytic agents for use in methods forselective killing of senescent cells are not intended to be used as atreatment for cancer, that is, in a manner that kills or destroys thecancer cells in a statistically significant manner. Therefore, themethods disclosed herein do not encompass use of the senolytic agents ina manner that would be considered a primary therapy for the treatment ofa cancer. Even though a senolytic agent, alone or with otherchemotherapeutic or radiotherapy agents, are not used in a manner thatis sufficient to be considered as a primary cancer therapy, the methodsand senolytic agents described herein may be used in a manner (e.g., ashort term course of therapy) that is useful for inhibiting metastases.In other certain embodiments, the subject to be treated with thesenolytic agent does not have a cancer (i.e., the subject has not beendiagnosed as having a cancer by a person skilled in the medical art).

Cardiovascular Diseases and Disorders.

In another embodiment, the senescence-associated disease or disordertreated by the methods described herein is a cardiovascular disease. Thecardiovascular disease may be any one or more of angina, arrhythmia,atherosclerosis, cardiomyopathy, congestive heart failure, coronaryartery disease (CAD), carotid artery disease, endocarditis, heart attack(coronary thrombosis, myocardial infarction [MI]), high bloodpressure/hypertension, aortic aneurysm, brain aneurysm, cardiacfibrosis, cardiac diastolic dysfunction,hypercholesterolemia/hyperlipidemia, mitral valve prolapse, peripheralvascular disease (e.g., peripheral artery disease (PAD)), cardiac stressresistance, and stroke.

In certain embodiments, methods are provided for treatingsenescence-associated cardiovascular disease that is associated with orcaused by arteriosclerosis (i.e., hardening of the arteries). Thecardiovascular disease may be any one or more of atherosclerosis (e.g.,coronary artery disease (CAD) and carotid artery disease); angina,congestive heart failure, and peripheral vascular disease (e.g.,peripheral artery disease (PAD)). The methods for treating acardiovascular disease that is associated with or caused byarteriosclerosis may reduce the likelihood of occurrence of high bloodpressure/hypertension, angina, stroke, and heart attack (i.e., coronarythrombosis, myocardial infarction (MI)). In certain embodiments, methodsare provided for stabilizing atherosclerotic plaque(s) in a blood vessel(e.g., artery) of a subject, thereby reducing the likelihood ofoccurrence or delaying the occurrence of a thrombotic event, such asstroke or MI. In certain embodiments, these methods comprisingadministration of a senolytic agent reduce (i.e., cause decrease of) thelipid content of an atherosclerotic plaque in a blood vessel (e.g.,artery) of the subject and/or increase the fibrous cap thickness (i.e.,cause an increase, enhance or promote thickening of the fibrous cap).

Atherosclerosis is characterized by patchy intimal plaques (atheromas)that encroach on the lumen of medium-sized and large arteries; theplaques contain lipids, inflammatory cells, smooth muscle cells, andconnective tissue. Atherosclerosis can affect large and medium-sizedarteries, including the coronary, carotid, and cerebral arteries, theaorta and its branches, and major arteries of the extremities.Atherosclerosis is characterized by patchy intimal plaques (atheromas)that encroach on the lumen of medium-sized and large arteries; theplaques contain lipids, inflammatory cells, smooth muscle cells, andconnective tissue.

In one embodiment, methods are provided for inhibiting the formation ofatherosclerotic plaques (or reducing, diminishing, causing decrease information of atherosclerotic plaques) by administering a senolyticagent. In other embodiments, methods are provided for reducing(decreasing, diminishing) the amount (i.e., level) of plaque. Reductionin the amount of plaque in a blood vessel (e.g., artery) may bedetermined, for example, by a decrease in surface area of the plaque, orby a decrease in the extent or degree (e.g., percent) of occlusion of ablood vessel (e.g., artery), which can be determined by angiography orother visualizing methods used in the cardiovascular art. Also providedherein are methods for increasing the stability (or improving,promoting, enhancing stability) of atherosclerotic plaques that arepresent in one or more blood vessels (e.g., one or more arteries) of asubject, which methods comprise administering to the subject any one ofthe senolytic agents described herein.

Atherosclerosis is often referred to as a “hardening” or furring of thearteries and is caused by the formation of multiple atheromatous plaqueswithin the arteries. Atherosclerosis (also called arterioscleroticvascular disease or ASVD herein and in the art) is a form ofarteriosclerosis in which an artery wall thickens. Symptoms develop whengrowth or rupture of the plaque reduces or obstructs blood flow; and thesymptoms may vary depending on which artery is affected. Atheroscleroticplaques may be stable or unstable. Stable plaques regress, remainstatic, or grow slowly, sometimes over several decades, until they maycause stenosis or occlusion. Unstable plaques are vulnerable tospontaneous erosion, fissure, or rupture, causing acute thrombosis,occlusion, and infarction long before they cause hemodynamicallysignificant stenosis. Most clinical events result from unstable plaques,which do not appear severe on angiography; thus, plaque stabilizationmay be a way to reduce morbidity and mortality. Plaque rupture orerosion can lead to major cardiovascular events such as acute coronarysyndrome and stroke (see, e.g., Du et al., BMC Cardiovascular Disorders14:83 (2014); Grimm et al., Journal of Cardiovascular Magnetic Resonance14:80 (2012)). Disrupted plaques were found to have a greater content oflipid, macrophages, and had a thinner fibrous cap than intact plaques(see, e.g., Felton et al., Arteriosclerosis, Thrombosis, and VascularBiology 17:1337-45 (1997)).

Atherosclerosis is a syndrome affecting arterial blood vessels due insignificant part to a chronic inflammatory response of white blood cellsin the walls of arteries. This is promoted by low-density lipoproteins(LDL, plasma proteins that carry cholesterol and triglycerides) in theabsence of adequate removal of fats and cholesterol from macrophages byfunctional high-density lipoproteins (HDL). The earliest visible lesionof atherosclerosis is the “fatty streak,” which is an accumulation oflipid-laden foam cells in the intimal layer of the artery. The hallmarkof atherosclerosis is atherosclerotic plaque, which is an evolution ofthe fatty streak and has three major components: lipids (e.g.,cholesterol and triglycerides); inflammatory cells and smooth musclecells; and a connective tissue matrix that may contain thrombi invarious stages of organization and calcium deposits. Within theouter-most and oldest plaque, calcium and other crystallized components(e.g., microcalcification) from dead cells can be found.Microcalcification and properties related thereto are also thought tocontribute to plaque instability by increasing plaque stress (see, e.g.,Bluestein et al., J. Biomech. 41 (5):1111-18 (2008); Cilia et al.,Journal of Engineering in Medicine 227:588-99 (2013)). Fatty streaksreduce the elasticity of the artery walls, but may not affect blood flowfor years because the artery muscular wall accommodates by enlarging atthe locations of plaque. Lipid-rich atheromas are at increased risk forplaque rupture and thrombosis (see, e.g., Felton et al., supra; Fusteret al., J. Am. Coll. Cardiol. 46:1209-18 (2005)). Reports have foundthat of all plaque components, the lipid core exhibits the highestthrombogenic activity (see, e.g., Femandez-Ortiz et al., J. Am. Coil.Cardiol. 23:1562-69 (1994)). Within major arteries in advanced disease,the wall stiffening may also eventually increase pulse pressure.

A vulnerable plaque that may lead to a thrombotic event (stroke or MI)and is sometimes described as a large, soft lipid pool covered by a thinfibrous cap (see, e.g., Li et al., Stroke 37:1195-99 (2006); Trivedi etal., Neuroradiology 46:738-43 (2004)). An advanced characteristicfeature of advance atherosclerotic plaque is irregular thickening of thearterial intima by inflammatory cells, extracellular lipid (atheroma)and fibrous tissue (sclerosis) (see, e.g., Newby et al., Cardiovasc.Res. 345-60 (1999)). Fibrous cap formation is believe to occur from themigration and proliferation of vascular smooth muscle cells and frommatrix deposition (see, e.g., Ross, Nature 362:801-809 (1993); Sullivanet al., J. Angiology at dx.doi.org/10.1155/2013/592815 (2013)). A thinfibrous cap contributes instability of the plaque and to increased riskfor rupture (see, e.g., Li et al., supra).

Both proinflammatory macrophages (M1) and anti-inflammatory macrophages(M2) can be found in arteriosclerotic plaque. The contribution of bothtypes to plaque instability is a subject of active investigation, withresults suggesting that an increased level of the M1 type versus the M2type correlates with increased instability of plaque (see, e.g., Medburyet al., Int. Angiol. 32:74-84 (2013); Lee et al., Am. J. Clin. Pathol.139:317-22 (2013); Martinet et al., Cir. Res. 751-53 (2007)).

Subjects suffering from cardiovascular disease can be identified usingstandard diagnostic methods known in the art for cardiovascular disease.Generally, diagnosis of atherosclerosis and other cardiovascular diseaseis based on symptoms (e.g., chest pain or pressure (angina), numbness orweakness in arms or legs, difficulty speaking or slurred speech,drooping muscles in face, leg pain, high blood pressure, kidney failureand/or erectile dysfunction), medical history, and/or physicalexamination of a patient. Diagnosis may be confirmed by angiography,ultrasonography, or other imaging tests. Subjects at risk of developingcardiovascular disease include those having any one or more ofpredisposing factors, such as a family history of cardiovascular diseaseand those having other risk factors (i.e., predisposing factors) such ashigh blood pressure, dyslipidemia, high cholesterol, diabetes, obesityand cigarette smoking, sedentary lifestyle, and hypertension. In acertain embodiment, the cardiovascular disease that is a senescence cellassociated disease/disorder is atherosclerosis.

The effectiveness of one or more senolytic agents for treating orpreventing (i.e., reducing or decreasing the likelihood of developing oroccurrence of) a cardiovascular disease (e.g., atherosclerosis) canreadily be determined by a person skilled in the medical and clinicalarts. One or any combination of diagnostic methods, including physicalexamination, assessment and monitoring of clinical symptoms, andperformance of analytical tests and methods described herein andpracticed in the art (e.g., angiography, electrocardiography, stresstest, non-stress test), may be used for monitoring the health status ofthe subject. The effects of the treatment of a senolytic agent orpharmaceutical composition comprising same can be analyzed usingtechniques known in the art, such as comparing symptoms of patientssuffering from or at risk of cardiovascular disease that have receivedthe treatment with those of patients without such a treatment or withplacebo treatment.

Inflammatory and Autoimmune Diseases and Disorders.

In certain embodiments, a senescence-associated disease or disorder isan inflammatory disease or disorder, such as by way of non-limitingexample, osteoarthritis, that may be treated or prevented (i.e.,likelihood of occurrence is reduced) according to the methods describedherein that comprise administration of a senolytic agent. Otherinflammatory or autoimmune diseases or disorders that may be treated byadministering a senolytic agent such as the inhibitors and antagonistsdescribed herein include osteoporosis, psoriasis, oral mucositis,rheumatoid arthritis, inflammatory bowel disease, eczema, kyphosis,herniated intervertebral disc, and the pulmonary diseases, COPD andidiopathic pulmonary fibrosis.

Osteoarthritis degenerative joint disease is characterized byfibrillation of the cartilage at sites of high mechanical stress, bonesclerosis, and thickening of the synovium and the joint capsule.Fibrillation is a local surface disorganization involving splitting ofthe superficial layers of the cartilage. The early splitting istangential with the cartilage surface, following the axes of thepredominant collagen bundles. Collagen within the cartilage becomesdisorganized, and proteoglycans are lost from the cartilage surface. Inthe absence of protective and lubricating effects of proteoglycans in ajoint, collagen fibers become susceptible to degradation, and mechanicaldestruction ensues. Predisposing risk factors for developingosteoarthritis include increasing age, obesity, previous joint injury,overuse of the joint, weak thigh muscles, and genetics. It is a commoncause of chronic disability in the elderly. Symptoms of osteoarthritisinclude sore or stiff joints, particularly the hips, knees, and lowerback, after inactivity or overuse; stiffness after resting that goesaway after movement; and pain that is worse after activity or toward theend of the day. Osteoarthritis may also affect the neck, small fingerjoints, the base of the thumb, ankle, and big toe.

Chronic inflammation is thought to be the main age-related factor thatcontributes to osteoarthritis. In combination with aging, joint overuseand obesity appear to promote osteoarthritis.

Unexpectedly, by selectively killing senescent cells a senolytic agentprevents (i.e., reduces the likelihood of occurrence), reduces orinhibits loss or erosion of proteoglycan layers in a joint, reducesinflammation in the affected joint, and promotes (i.e., stimulates,enhances, induces) production of collagen (e.g., type 2 collagen).Removal of senescent cells causes a reduction in the amount (i.e.,level) of inflammatory cytokines, such as IL-6, produced in a joint andinflammation is reduced. Methods are provided herein for treatingosteoarthritis, for selectively killing senescent cells in anosteoarthritic joint of a subject, and/or inducing collagen (such asType 2 collagen) production in the joint of a subject in need thereof byadministering at least one senolytic agent (which may be combined withat least one pharmaceutically acceptable excipient to form apharmaceutical composition) to the subject. A senolytic agent also maybe used for decreasing (inhibiting, reducing) production ofmetalloproteinase 13 (MMP-13), which degrades collagen in a joint, andfor restoring proteoglycan layer or inhibiting loss and/or degradationof the proteoglycan layer. Treatment with the senolytic agent therebyalso prevents (i.e., reduces likelihood of occurrence of), inhibits, ordecreases erosion, or slows (i.e., decreases rate) erosion of the bone.As described in detail herein, in certain embodiments, the senolyticagent is administered directly to an osteoarthritic joint (e.g., byintra-articularly, topical, transdermal, intradermal, or subcutaneousdelivery). Treatment with a senolytic agent can also restore, improve,or inhibit deterioration of strength of a joint. In addition, themethods comprising administering a senolytic agent can reduce joint painand are therefore useful for pain management of osteoarthritic joints.

The effectiveness of one or more senolytic agents for treatment orprophylaxis of osteoarthritis in a subject and monitoring of a subjectwho receives one or more senolytic agents can readily be determined by aperson skilled in the medical and clinical arts. One or any combinationof diagnostic methods, including physical examination (such asdetermining tenderness, swelling or redness of the affected joint),assessment and monitoring of clinical symptoms (such as pain, stiffness,mobility), and performance of analytical tests and methods describedherein and practiced in the art (e.g., determining the level ofinflammatory cytokines or chemokines; X-ray images to determine loss ofcartilage as shown by a narrowing of space between the bones in a joint;magnetic resonance imaging (MRI), providing detailed images of bone andsoft tissues, including cartilage), may be used for monitoring thehealth status of the subject. The effects of the treatment of one ormore senolytic agents can be analyzed by comparing symptoms of patientssuffering from or at risk of an inflammatory disease or disorder, suchas osteoarthritis, who have received the treatment with those ofpatients who have not received such a treatment or who have received aplacebo treatment.

In certain embodiments, senolytic agents may be used for treating and/orpreventing (i.e., decreasing or reducing the likelihood of occurrence)rheumatoid arthritis (RA). Dysregulation of innate and adaptive immuneresponses characterize rheumatoid arthritis (RA), which is an autoimmunedisease the incidence of which increases with age. Rheumatoid arthritisis a chronic inflammatory disorder that typically affects the smalljoints in hands and feet. Whereas osteoarthritis results from, at leastin part, wear and tear of a joint, rheumatoid arthritis affects thelining of joints, resulting in a painful swelling that can lead to boneerosion and joint deformity. RA can sometimes also affect other organsof the body, such as the skin, eyes, lungs and blood vessels. RA canoccur in a subject at any age; however, RA usually begins to developafter age 40. The disorder is much more common in women. In certainembodiments of the methods described herein, RA is excluded.

Chronic inflammation may also contribute to other age-related or agingrelated diseases and disorders, such as kyphosis and osteoporosis.Kyphosis is a severe curvature in the spinal column, and it isfrequently seen with normal and premature aging (see, e.g., Katzman etal. (2010) J. Orthop. Sports Phys. Ther. 40: 352-360). Age-relatedkyphosis often occurs after osteoporosis weakens spinal bones to thepoint that they crack and compress. A few types of kyphosis targetinfants or teens. Severe kyphosis can affect lungs, nerves, and othertissues and organs, causing pain and other problems. Kyphosis has beenassociated with cellular senescence. Characterizing the capability of asenolytic agent for treating kyphosis may be determined in pre-clinicalanimal models used in the art. By way of example, TTD mice developkyphosis (see, e.g., de Boer et al. (2002) Science 296: 1276-1279);other mice that may be used include BubR1^(H/H) mice, which are alsoknown to develop kyphosis (see, e.g., Baker et al. (2011) Nature 479:232-36). Kyphosis formation is visually measured over time. The level ofsenescent cells decreased by treatment with the senolytic agent can bedetermined by detecting the presence of one or more senescent cellassociated markers such as by SA-β-GAL staining.

Osteoporosis is a progressive bone disease that is characterized by adecrease in bone mass and density that may lead to an increased risk offracture. Bone mineral density (BMD) is reduced, bone microarchitecturedeteriorates, and the amount and variety of proteins in bone arealtered. Osteoporosis is typically diagnosed and monitored by a bonemineral density test. Post-menopausal women or women who have reducedestrogen are most at risk. While both men and women over 75 are at risk,women are twice as likely to develop osteoporosis than men. The level ofsenescent cells decreased by treatment with the senolytic agent can bedetermined by detecting the presence of one or more senescent cellassociated markers such as by SA-β-GAL staining.

In still other embodiments, an inflammatory/autoimmune disorder that maybe treated or prevented (i.e., likelihood of occurrence is reduced) withthe senolytic agents described herein includes irritable bowel syndrome(IBS) and inflammatory bowel diseases, such as ulcerative colitis andCrohn's disease. Inflammatory bowel disease (IBD) involves chronicinflammation of all or part of the digestive tract. In addition tolife-threatening complications arising from IBD, the disease can bepainful and debilitating. Ulcerative colitis is an inflammatory boweldisease that causes long-lasting inflammation in part of the digestivetract. Symptoms usually develop over time, rather than suddenly.Ulcerative colitis usually affects only the innermost lining of thelarge intestine (colon) and rectum. Crohn's disease is an inflammatorybowel disease that causes inflammation anywhere along the lining of yourdigestive tract, and often extends deep into affected tissues. This canlead to abdominal pain, severe diarrhea, and malnutrition. Theinflammation caused by Crohn's disease can involve different areas ofthe digestive tract. Diagnosis and monitoring of the diseases isperformed according to methods and diagnostic tests routinely practicedin the art, including blood tests, colonoscopy, flexible sigmoidoscopy,barium enema, CT scan, MRI, endoscopy, and small intestine imaging.

In other embodiments, the methods described herein may be useful fortreating a subject who has herniated intervertebral discs. Subjects withthese herniated discs exhibit elevated presence of cell senescence inthe blood and in vessel walls (see e.g., Roberts et al. (2006) Eur.Spine J. 15 Suppl 3: S312-316). Symptoms of a herniated intervertebraldisc may include pain, numbness or tingling, or weakness in an arm orleg. Increased levels of proinflammatory molecules and matrixmetalloproteases are also found in aging and degenerating discs tissues,suggesting a role for senescence cells (see e.g., Chang-Qing et al.(2007) Ageing Res. Rev. 6: 247-61). Animal models may be used tocharacterize the effectiveness of a senolytic agent in treatingherniated intervertebral discs; degeneration of the intervertebral discis induced in mice by compression and disc strength evaluated (see e.g.,Lotz et al. (1998) Spine (Philadelphia Pa. 1976). 23:2493-506).

Other inflammatory or autoimmune diseases that may be treated orprevented (i.e., likelihood of occurrence is reduced) by using asenolytic agent include eczema, psoriasis, osteoporosis, and pulmonarydiseases (e.g., chronic obstructive pulmonary disease (COPD), idiopathicpulmonary fibrosis (IPF), asthma), inflammatory bowel disease, andmucositis (including oral mucositis, which in some instances is inducedby radiation). Certain fibrosis or fibrotic conditions of organs such asrenal fibrosis, liver fibrosis, pancreatic fibrosis, cardiac fibrosis,skin wound healing, and oral submucous fibrosis may be treated withusing the senolytic agent.

In certain embodiments, the senescent cell associated disorder is aninflammatory disorder of the skin, such as by way of a non-limitingexamples, psoriasis and eczema that may be treated or prevented (i.e.,likelihood of occurrence is reduced) according to the methods describedherein that comprise administration of a senolytic agent. Psoriasis ischaracterized by abnormally excessive and rapid growth of the epidermallayer of the skin. A diagnosis of psoriasis is usually based on theappearance of the skin. Skin characteristics typical for psoriasis arescaly red plaques, papules, or patches of skin that may be painful anditch. In psoriasis, cutaneous and systemic overexpression of variousproinflammatory cytokines is observed such as IL-6, a key component ofthe SASP. Eczema is an inflammation of the skin that is characterized byredness, skin swelling, itching and dryness, crusting, flaking,blistering, cracking, oozing, or bleeding. The effectiveness ofsenolytic agents for treatment of psoriasis and eczema and monitoring ofa subject who receives such a senolytic agent can be readily determinedby a person skilled in the medical or clinical arts. One or anycombination of diagnostic methods, including physical examination (suchas skin appearance), assessment of monitoring of clinical symptoms (suchas itching, swelling, and pain), and performance of analytical tests andmethods described herein and practiced in the art (i.e., determining thelevel of pro-inflammatory cytokines).

Other immune disorders or conditions that may be treated or prevented(i.e., likelihood of occurrence is reduced) with a senolytic agentinclude conditions resulting from a host immune response to an organtransplant (e.g., kidney, bone marrow, liver, lung, or hearttransplant), such as rejection of the transplanted organ. The senolyticagent may be used for treating or reducing the likelihood of occurrenceof graft-vs-host disease.

Pulmonary Diseases and Disorders.

In one embodiment, methods are provided for treating ore preventing(i.e., reducing the likelihood of occurrence of) a senescence-associateddisease or disorder that is a pulmonary disease or disorder by killingsenescent cells (i.e., established senescent cells) associated with thedisease or disorder in a subject who has the disease or disorder byadministering a senolytic agent. Senescence associated pulmonarydiseases and disorders include, for example, idiopathic pulmonaryfibrosis (IPF), chronic obstructive pulmonary disease (COPD), asthma,cystic fibrosis, bronchiectasis, and emphysema.

COPD is a lung disease defined by persistently poor airflow resultingfrom the breakdown of lung tissue (emphysema) and the dysfunction of thesmall airways (obstructive bronchiolitis). Primary symptoms of COPDinclude shortness of breath, wheezing, chest tightness, chronic cough,and excess sputum production. Elastase from cigarette smoke-activatedneutrophils and macrophages disintegrates the extracellular matrix ofalveolar structures, resulting in enlarged air spaces and loss ofrespiratory capacity (see, e.g., Shapiro et al., Am. J. Respir. CellMol. Biol. 32, 367-372 (2005)). COPD is most commonly caused by tobaccosmoke (including cigarette smoke, cigar smoke, secondhand smoke, pipesmoke), occupational exposure (e.g., exposure to dust, smoke or fumes),and pollution, occurring over decades thereby implicating aging as arisk factor for developing COPD.

The processes involved in causing lung damage include, for example,oxidative stress produced by the high concentrations of free radicals intobacco smoke; cytokine release due to inflammatory response toirritants in the airway; and impairment of anti-protease enzymes bytobacco smoke and free radicals, allowing proteases to damage the lungs.Genetic susceptibility can also contribute to the disease. In about 1%percent of people with COPD, the disease results from a genetic disorderthat causes low level production of alpha-1-antitrypsin in the liver.The enzyme is normally secreted into the bloodstream to help protect thelungs.

Pulmonary fibrosis is a chronic and progressive lung diseasecharacterized by stiffening and scarring of the lung, which may lead torespiratory failure, lung cancer, and heart failure. Fibrosis isassociated with repair of epithelium. Fibroblasts are activated,production of extracellular matrix proteins is increased, andtransdifferentiation to contractile myofibroblasts contribute to woundcontraction. A provisional matrix plugs the injured epithelium andprovides a scaffold for epithelial cell migration, involving anepithelial-mesenchymal transition (EMT). Blood loss associated withepithelial injury induces platelet activation, production of growthfactors, and an acute inflammatory response. Normally, the epithelialbarrier heals and the inflammatory response resolves. However, infibrotic disease the fibroblast response continues, resulting inunresolved wound healing. Formation of fibroblastic foci is a feature ofthe disease, reflecting locations of ongoing fibrogenesis. As the nameconnotes, the etiology of IPF is unknown. The involvement of cellularsenescence in IPF is suggested by the observations that the incidence ofthe disease increases with age and that lung tissue in IPF patients isenriched for SA-β-Gal-positive cells and contains elevated levels of thesenescence marker p21 (see, e.g., Minagawa et al., Am. J. Physiol. LungCell. Mol. Physiol. 300:L391-L401 (2011); see also, e.g., Naylor et al.,supra). Short telomeres are a risk factor common to both IPF andcellular senescence (see, e.g., Alder et al., Proc. Natl. Acad. Sci. USA105:13051-56 (2008)). Without wishing to be bound by theory, thecontribution of cellular senescence to IPF is suggested by the reportthat SASP components of senescent cells, such as IL-6, IL-8, and IL-13,promote fibroblast-to-myofibroblast differentiation andepithelial-mesenchymal transition, resulting in extensive remodeling ofthe extracellular matrix of the alveolar and interstitial spaces (see,e.g., Minagawa et al., supra).

Subjects at risk of developing pulmonary fibrosis include those exposedto environmental or occupational pollutants, such as asbestosis andsilicosis; who smoke cigarettes; having some typical connective tissuediseases such as rheumatoid arthritis, SLE and scleroderma; having otherdiseases that involve connective tissue, such as sarcoidosis andWegener's granulomatosis; having infections; taking certain medications(e.g., amiodarone, bleomycin, busufan, methotrexate, andnitrofurantoin); those subject to radiation therapy to the chest; andthose whose family member has pulmonary fibrosis.

Symptoms of COPD may include any one of shortness of breath, especiallyduring physical activities; wheezing; chest tightness; having to clearyour throat first thing in the morning because of excess mucus in thelungs; a chronic cough that produces sputum that may be clear, white,yellow or greenish; blueness of the lips or fingernail beds (cyanosis);frequent respiratory infections; lack of energy; unintended weight loss(observed in later stages of disease). Subjects with COPD may alsoexperience exacerbations, during which symptoms worsen and persist fordays or longer. Symptoms of pulmonary fibrosis are known in the art andinclude shortness of breath, particularly during exercise; dry, hackingcough; fast, shallow breathing; gradual unintended weight loss;tiredness; aching joints and muscles; and clubbing (widening androunding of the tips of the fingers or toes).

Subjects suffering from COPD or pulmonary fibrosis can be identifiedusing standard diagnostic methods routinely practiced in the art.Monitoring the effect of one or more senolytic agents administered to asubject who has or who is at risk of developing a pulmonary disease maybe performed using the methods typically used for diagnosis. Generally,one or more of the following exams or tests may be performed: physicalexam, patient's medical history, patient's family's medical history,chest X-ray, lung function tests (such as spirometry), blood test (e.g.,arterial blood gas analysis), bronchoalveolar lavage, lung biopsy, CTscan, and exercise testing.

Other pulmonary diseases or disorders that may be treated by using asenolytic agent include, for example, emphysema, asthma, bronchiectasis,and cystic fibrosis (see, e.g., Fischer et al., Am J Physiol Lung CellMol Physiol. 304(6):L394-400 (2013)). These diseases may also beexacerbated by tobacco smoke (including cigarette smoke, cigar smoke,secondhand smoke, pipe smoke), occupational exposure (e.g., exposure todust, smoke or fumes), infection, and/or pollutants that induce cellsinto senescence and thereby contribute to inflammation. Emphysema issometimes considered as a subgroup of COPD.

Bronchiectasis is results from damage to the airways that causes them towiden and become flabby and scarred. Bronchiectasis usually is caused bya medical condition that injures the airway walls or inhibits theairways from clearing mucus. Examples of such conditions include cysticfibrosis and primary ciliary dyskinesia (PCD). When only one part of thelung is affected, the disorder may be caused by a blockage rather than amedical condition.

The methods described herein for treating or preventing (i.e., reducingthe likelihood of occurrence of) a senescence associate pulmonarydisease or disorder may also be used for treating a subject who is agingand has loss (or degeneration) of pulmonary function (i.e., declining orimpaired pulmonary function compared with a younger subject) and/ordegeneration of pulmonary tissue. The respiratory system undergoesvarious anatomical, physiological and immunological changes with age.The structural changes include chest wall and thoracic spine deformitiesthat can impair the total respiratory system compliance resulting inincreased effort to breathe. The respiratory system undergoesstructural, physiological, and immunological changes with age. Anincreased proportion of neutrophils and lower percentage of macrophagescan be found in bronchoalveolar lavage (BAL) of older adults comparedwith younger adults. Persistent low grade inflammation in the lowerrespiratory tract can cause proteolytic and oxidant-mediated injury tothe lung matrix resulting in loss of alveolar unit and impaired gasexchange across the alveolar membrane seen with aging. Sustainedinflammation of the lower respiratory tract may predispose older adultsto increased susceptibility to toxic environmental exposure andaccelerated lung function decline. (See, for example, Sharma et al.,Clinical Interventions in Aging 1:253-60 (2006)). Oxidative stressexacerbates inflammation during aging (see, e.g., Brod, Inflamm Res2000; 49:561-570; Hendel et al., Cell Death and Differentiation (2010)17:596-606). Alterations in redox balance and increased oxidative stressduring aging precipitate the expression of cytokines, chemokines, andadhesion molecules, and enzymes (see, e.g., Chung et al., Ageing Res Rev2009; 8:18-30). Constitutive activation and recruitment of macrophages,T cells, and mast cells foster release of proteases leading toextracellular matrix degradation, cell death, remodeling, and otherevents that can cause tissue and organ damage during chronicinflammation (see, e.g., Demedts et al., Respir Res 2006; 7: 53-63). Byadministering a senolytic agent to an aging subject (which includes amiddle-aged adult who is asymptomatic), the decline in pulmonaryfunction may be decelerated or inhibited by killing and removingsenescent cells from the respiratory tract.

The effectiveness of a senolytic agent can readily be determined by aperson skilled in the medical and clinical arts. One or any combinationof diagnostic methods, including physical examination, assessment andmonitoring of clinical symptoms, and performance of analytical tests andmethods described herein, may be used for monitoring the health statusof the subject. The effects of the treatment of a senolytic agent orpharmaceutical composition comprising the agent can be analyzed usingtechniques known in the art, such as comparing symptoms of patientssuffering from or at risk of the pulmonary disease that have receivedthe treatment with those of patients without such a treatment or withplacebo treatment. In addition, methods and techniques that evaluatemechanical functioning of the lung, for example, techniques that measurelung capacitance, elastance, and airway hypersensitivity may beperformed. To determine lung function and to monitor lung functionthroughout treatment, any one of numerous measurements may be obtained,expiratory reserve volume (ERV), forced vital capacity (FVC), forcedexpiratory volume (FEV) (e.g., FEV in one second, FEV1), FEV1/FEV ratio,forced expiratory flow 25% to 75%, and maximum voluntary ventilation(MVV), peak expiratory flow (PEF), slow vital capacity (SVC). Total lungvolumes include total lung capacity (TLC), vital capacity (VC), residualvolume (RV), and functional residual capacity (FRC). Gas exchange acrossalveolar capillary membrane can be measured using diffusion capacity forcarbon monoxide (DLCO). Peripheral capillary oxygen saturation (SpO₂)can also be measured; normal oxygen levels are typically between 95% and100%. An SpO₂ level below 90% suggests the subject has hypoxemia. Valuesbelow 80% are considered critical and requiring intervention to maintainbrain and cardiac function and avoid cardiac or respiratory arrest.

Neurological Diseases and Disorders.

Senescence-associated diseases or disorders treatable by administering asenolytic agent described herein include neurological diseases ordisorders. Such senescence-associated diseases and disorders includeParkinson's disease, Alzheimer's disease (and other dementias), motorneuron dysfunction (MND), mild cognitive impairment (MCI), Huntington'sdisease, and diseases and disorders of the eyes, such as age-relatedmacular degeneration. Other diseases of the eye that are associated withincreasing age are glaucoma, vision loss, presbyopia, and cataracts.

Parkinson's disease (PD) is the second most common neurodegenerativedisease. It is a disabling condition of the brain characterized byslowness of movement (bradykinesia), shaking, stiffness, and in thelater stages, loss of balance. Many of these symptoms are due to theloss of certain nerves in the brain, which results in the lack ofdopamine. This disease is characterized by neurodegeneration, such asthe loss of about 50% to 70% of the dopaminergic neurons in thesubstantia nigra pars compacta, a profound loss of dopamine in thestriatum, and/or the presence of intracytoplasmic inclusions (Lewybodies), which are composed mainly of alpha-synuclein and ubiquitin.Parkinson's disease also features locomotor deficits, such as tremor,rigidity, bradykinesia, and/or postural instability. Subjects at risk ofdeveloping Parkinson's disease include those having a family history ofParkinson's disease and those exposed to pesticides (e.g., rotenone orparaquat), herbicides (e.g., agent orange), or heavy metals. Senescenceof dopamine-producing neurons is thought to contribute to the observedcell death in PD through the production of reactive oxygen species (see,e.g., Cohen et al., J. Neural Transm. Suppl. 19:89-103 (1983));therefore, the methods and senolytic agents described herein are usefulfor treatment and prophylaxis of Parkinson's disease.

Methods for detecting, monitoring or quantifying neurodegenerativedeficiencies and/or locomotor deficits associated with Parkinson'sdiseases are known in the art, such as histological studies, biochemicalstudies, and behavioral assessment (see, e.g., U.S. ApplicationPublication No. 2012/0005765). Symptoms of Parkinson's disease are knownin the art and include, but are not limited to, difficulty starting orfinishing voluntary movements, jerky, stiff movements, muscle atrophy,shaking (tremors), and changes in heart rate, but normal reflexes,bradykinesia, and postural instability. There is a growing recognitionthat people diagnosed with Parkinson's disease may have cognitiveimpairment, including mild cognitive impairment, in addition to theirphysical symptoms.

Alzheimer's disease (AD) is a neurodegenerative disease that shows aslowly progressive mental deterioration with failure of memory,disorientation, and confusion, leading to profound dementia. Age is thesingle greatest predisposing risk factor for developing AD, which is theleading cause of dementia in the elderly (see, e.g., Hebert, et al.,Arch. Neurol. 60:1119-1122 (2003)). Early clinical symptoms showremarkable similarity to mild cognitive impairment (see below). As thedisease progresses, impaired judgment, confusion, behavioral changes,disorientation, and difficulty in walking and swallowing occur.

Alzheimer's disease is characterized by the presence of neurofibrillarytangles and amyloid (senile) plaques in histological specimens. Thedisease predominantly involves the limbic and cortical regions of thebrain. The argyrophilic plaques containing the amyloidogenic Aβ fragmentof amyloid precursor protein (APP) are scattered throughout the cerebralcortex and hippocampus. Neurofibrillary tangles are found in pyramidalneurons predominantly located in the neocortex, hippocampus, and nucleusbasalis of Meynert. Other changes, such as granulovacuolar degenerationin the pyramidal cells of the hippocampus, and neuron loss and gliosisin the cortex and hippocampus, are observed. Subjects at risk ofdeveloping Alzheimer's disease include those of advanced age, those witha family history of Alzheimer's disease, those with genetic risk genes(e.g., ApoE4) or deterministic gene mutations (e.g., APP, PS1, or PS2),and those with history of head trauma or heart/vascular conditions(e.g., high blood pressure, heart disease, stroke, diabetes, highcholesterol).

A number of behavioral and histopathological assays are known in the artfor evaluating Alzheimer's disease phenotype, for characterizingtherapeutic agents, and assessing treatment. Histological analyses aretypically performed postmortem. Histological analysis of A1 levels maybe performed using Thioflavin-S. Congo red, or anti-Aβ staining (e.g.,4G8, 10D5, or 6E10 antibodies) to visualize AP deposition on sectionedbrain tissues (see, e.g., Holcomb et al., 1998, Nat. Med. 4:97-100;Borchelt et al., 1997, Neuron 19:939-945; Dickson et al., 1988, Am. J.Path. 132:86-101). In vivo methods of visualizing A3 deposition intransgenic mice have been also described. BSB ((trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene) andPET tracer ¹¹C-labelled Pittsburgh Compound-B (PIB) bind to Aβ plaques(see, e.g., Skovronsky et al., 2000, Proc. Natl. Acad. Sci. USA97:7609-7614; Klunk et al., 2004, Ann. Neurol. 55:306-319).¹⁹F-containing amyloidophilic Congo red-type compound FSB((E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene)allows visualization of A1 plaques by MRI (see, e.g., Higuchi et al.,2005, Nature Neurosci. 8:527-533). Radiolabeled, putrescine-modifiedamyloid-beta peptide labels amyloid deposits in vivo in a mouse model ofAlzheimer's disease (see, e.g., Wengenack et al., 2000, Nat. Biotechnol.18:868-872).

Increased glial fibrillary acidic protein (GFAP) by astrocytes is amarker for astroglial activation and gliosis during neurodegeneration.A3 plaques are associated with GFAP-positive activated astrocytes, andmay be visualized via GFAP staining (see, e.g., Nagele et al. 2004,Neurobiol. Aging 25:663-674; Mandybur et al., 1990, Neurology40:635-639; Liang et al., 2010, J. Biol. Chem. 285:27737-27744).Neurofibrillary tangles may be identified by immunohistochemistry usingthioflavin-S fluorescent microscopy and Gallyas silver stains (see,e.g., Gotz et al., 2001, J. Biol. Chem. 276:529-534; U.S. Pat. No.6,664,443). Axon staining with electron microscopy and axonal transportstudies may be used to neuronal degeneration (see, e.g., Ishihara etal., 1999, Neuron 24:751-762).

Subjects suffering from Alzheimer's disease can be identified usingstandard diagnostic methods known in the art for Alzheimer's disease.Generally, diagnosis of Alzheimer's disease is based on symptoms (e.g.,progressive decline in memory function, gradual retreat from andfrustration with normal activities, apathy, agitation or irritability,aggression, anxiety, sleep disturbance, dysphoria, aberrant motorbehavior, disinhibition, social withdrawal, decreased appetite,hallucinations, dementia), medical history, neuropsychological tests,neurological and/or physical examination of a patient. Cerebrospinalfluid may also be for tested for various proteins that have beenassociated with Alzheimer pathology, including tau, amyloid betapeptide, and AD7C-NTP. Genetic testing is also available for early-onsetfamilial Alzheimer disease (eFAD), an autosomal-dominant geneticdisease. Clinical genetic testing is available for individuals with ADsymptoms or at-risk family members of patients with early-onset disease.In the U.S., mutations for PS2, and APP may be tested in a clinical orfederally approved laboratory under the Clinical Laboratory ImprovementAmendments. A commercial test for PS1 mutations is also available (ElanPharmaceuticals).

The effectiveness of one or more senolytic agents described herein andmonitoring of a subject who receives one or more senolytic agents canreadily be determined by a person skilled in the medical and clinicalarts. One or any combination of diagnostic methods, including physicalexamination, assessment and monitoring of clinical symptoms, andperformance of analytical tests and methods described herein, may beused for monitoring the health status of the subject. The effects ofadministering one or more senolytic agents can be analyzed usingtechniques known in the art, such as comparing symptoms of patientssuffering from or at risk of Alzheimer's disease that have received thetreatment with those of patients without such a treatment or withplacebo treatment.

Mild Cognitive Impairment (MCI).

MCI is a brain-function syndrome involving the onset and evolution ofcognitive impairments beyond those expected based on age and educationof the individual, but which are not significant enough to interferewith this individual's daily activities. MCI is an aspect of cognitiveaging that is considered to be a transitional state between normal agingand the dementia into which it may convert (see, Pepeu, Dialogues inClinical Neuroscience 6:369-377, 2004). MCI that primarily affectsmemory is known as “amnestic MCI.” A person with amnestic MCI may startto forget important information that he or she would previously haverecalled easily, such as recent events. Amnestic MCI is frequently seenas prodromal stage of Alzheimer's disease. MCI that affects thinkingskills other than memory is known as “non-amnestic MCI.” This type ofMCI affect thinking skills such as the ability to make sound decisions,judge the time or sequence of steps needed to complete a complex task,or visual perception. Individuals with non-amnestic MCI are believed tobe more likely to convert to other types of dementias (e.g., dementiawith Lewy bodies).

Persons in the medical art have a growing recognition that peoplediagnosed with Parkinson's disease may have MCI in addition to theirphysical symptoms. Recent studies show 20-30% of people with Parkinson'sdisease have MCI, and that their MCI tends to be non-amnestic.Parkinson's disease patients with MCI sometimes go on to develop fullblown dementia (Parkinson's disease with dementia).

Methods for detecting, monitoring, quantifying or assessingneuropathological deficiencies associated with MCI are known in the art,including astrocyte morphological analyses, release of acetylcholine,silver staining for assessing neurodegeneration, and PiB PET imaging todetect beta amyloid deposits (see, e.g., U.S. Application PublicationNo. 2012/0071468; Pepeu, 2004, supra). Methods for detecting,monitoring, quantifying or assessing behavioral deficiencies associatedwith MCI are also known in the art, including eight-arm radial mazeparadigm, non-matching-to-sample task, allocentric place determinationtask in a water maze, Morris maze test, visuospatial tasks, and delayedresponse spatial memory task, olfactory novelty test (see, id.).

Motor Neuron Dysfunction (MND).

MND is a group of progressive neurological disorders that destroy motorneurons, the cells that control essential voluntary muscle activity suchas speaking, walking, breathing and swallowing. It is classifiedaccording to whether degeneration affects upper motor neurons, lowermotor neurons, or both. Examples of MNDs include, but are not limited toAmyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's Disease,progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis,progressive muscular atrophy, lower motor neuron disease, and spinalmuscular atrophy (SMA) (e.g., SMA1 also called Werdnig-Hoffmann Disease,SMA2, SMA3 also called Kugelberg-Welander Disease, and Kennedy'sdisease), post-polio syndrome, and hereditary spastic paraplegia. Inadults, the most common MND is amyotrophic lateral sclerosis (ALS),which affects both upper and lower motor neurons. It can affect thearms, legs, or facial muscles. Primary lateral sclerosis is a disease ofthe upper motor neurons, while progressive muscular atrophy affects onlylower motor neurons in the spinal cord. In progressive bulbar palsy, thelowest motor neurons of the brain stem are most affected, causingslurred speech and difficulty chewing and swallowing. There are almostalways mildly abnormal signs in the arms and legs. Patients with MNDexhibit a phenotype of Parkinson's disease (e.g., having tremor,rigidity, bradykinesia, and/or postural instability). Methods fordetecting, monitoring or quantifying locomotor and/or other deficitsassociated with Parkinson's diseases, such as MND, are known in the art(see, e.g., U.S. Application Publication No. 20120005765).

Methods for detecting, monitoring, quantifying or assessing motordeficits and histopathological deficiencies associated with MND areknown in the art, including histopathological, biochemical, andelectrophysiological studies and motor activity analysis (see, e.g.,Rich et al., J Neurophysiol 88:3293-3304, 2002; Appel et al., Proc.Natl. Acad. Sci. USA 88:647-51, 1991). Histopathologically, MNDs arecharacterized by death of motor neurons, progressive accumulation ofdetergent-resistant aggregates containing SOD1 and ubiquitin andaberrant neurofilament accumulations in degenerating motor neurons. Inaddition, reactive astroglia and microglia are often detected indiseased tissue. Patients with an MND show one or more motor deficits,including muscle weakness and wasting, uncontrollable twitching,spasticity, slow and effortful movements, and overactive tendonreflexes.

Ophthalmic Diseases and Disorders:

In certain embodiments, a senescence-associated disease or disorder isan ocular disease, disorder, or condition, for example, presbyopia,macular degeneration, or cataracts. In other certain embodiments, thesenescence-associated disease or disorder is glaucoma. Maculardegeneration is a neurodegenerative disease that causes the loss ofphotoreceptor cells in the central part of retina, called the macula.Macular degeneration generally is classified into two types: dry typeand wet type. The dry form is more common than the wet, with about 90%of age-related macular degeneration (ARMD or AMD) patients diagnosedwith the dry form. The wet form of the disease usually leads to moreserious vision loss. While the exact causes of age-related maculardegeneration are still unknown, the number of senescent retinalpigmented epithelial (RPE) cells increases with age. Age and certaingenetic factors and environmental factors are risk factors fordeveloping ARMD (see, e.g., Lyengar et al., Am. J. Hum. Genet. 74:20-39(2004) (Epub 2003 Dec. 19); Kenealy et al., Mol. Vis. 10:57-61 (2004);Gorin et al., Mol. Vis. 5:29 (1999)). Environment predisposing factorsinclude omega-3 fatty acids intake (see, e.g., Christen et al., ArchOphthalmol. 129:921-29 (2011)); estrogen exposure (see, e.g., Feshanichet al., Arch Ophthalmol 126(4):519-24) (2008)); and increased serumlevels of vitamin D (see, e.g., Millen, et al., Arch Ophthalmol.129(4):481-89 (2011)). Genetic predisposing risk factors include reducedlevels Dicer1 (enzyme involved in maturation of micro RNA) in eyes ofpatients with dry AMD, and decreased micro RNAs contributes to asenescent cell profile; and DICER1 ablation induces premature senescence(see, e.g., Mudhasani J. Cell. Biol. (2008)).

Dry ARMD is associated with atrophy of RPE layer, which causes loss ofphotoreceptor cells. The dry form of ARMD may result from aging andthinning of macular tissues and from deposition of pigment in themacula. Senescence appears to inhibit both replication and migration ofRPE, resulting in permanent RPE depletion in the macula of dry AMDpatients (see, e.g., Iriyama et al., J. Biol. Chem. 283:11947-953(2008)). With wet ARMD, new blood vessels grow beneath the retina andleak blood and fluid. This abnormal leaky choroidal neovascularizationcauses the retinal cells to die, creating blind spots in central vision.Different forms of macular degeneration may also occur in youngerpatients. Non-age related etiology may be linked to heredity, diabetes,nutritional deficits, head injury, infection, or other factors.

Declining vision noticed by the patient or by an ophthalmologist duringa routine eye exam may be the first indicator of macular degeneration.The formation of exudates, or “drusen,” underneath the Bruch's membraneof the macula is often the first physical sign that macular degenerationmay develop. Symptoms include perceived distortion of straight linesand, in some cases, the center of vision appears more distorted than therest of a scene; a dark, blurry area or “white-out” appears in thecenter of vision; and/or color perception changes or diminishes.Diagnosing and monitoring of a subject with macular degeneration may beaccomplished by a person skilled in the ophthalmic art according toart-accepted periodic eye examination procedures and report of symptomsby the subject.

Presbyopia is an age-related condition where the eye exhibits aprogressively diminished ability to focus on near objects as the speedand amplitude of accommodation of a normal eye decreases with advancingage. Loss of elasticity of the crystalline lens and loss ofcontractility of the ciliary muscles have been postulated as its cause(see, e.g., Heys et al., 2004, Mol. Vis. 10:956-63; Petrash, 2013,Invest. Ophthalmol. Vis. Sci. 54:ORSF54-ORSF59). Age-related changes inthe mechanical properties of the anterior lens capsule and posteriorlens capsule suggest that the mechanical strength of the posterior lenscapsule decreases significantly with age (see, e.g., Krag et al.,Invest. Ophthalmol. Vis. Sci. 44:691-96 (2003); Krag et al., Invest.Ophthalmol. Vis. Sci. 38:357-63 (1997)).

The laminated structure of the capsule also changes and may result, atleast in part, from a change in the composition of the tissue (see,e.g., Krag et al., 1997, supra, and references cited therein). The majorstructural component of the lens capsule is basement membrane type IVcollagen that is organized into a three-dimensional molecular network(see, e.g., Cummings et al., Connect. Tissue Res. 55:8-12 (2014); Veiset al., Coll. Relat. Res. 1981; 1:269-86). Type IV collagen is composedof six homologous a chains (α1-6) that associate into heterotrimericcollagen IV protomers with each comprising a specific chain combinationof α112, α345, or α556 (see, e.g., Khoshnoodi et al., Microsc. Res.Tech. 2008; 71:357-70). Protomers share structural similarities of atriple-helical collagenous domain with the triplet peptide sequence ofGly-X-Y (Timpl et al., Eur. J. Biochem. 1979; 95:255-263), ending in aglobular C-terminal region termed the non-collagenous 1 (NC1) domain.The N-termini are composed of a helical domain termed the 7S domain(see, e.g., Risteli et al., Eur. J. Biochem. 1980; 108:239-250), whichis also involved in protomer-protomer interactions.

Research has suggested that collagen IV influences cellular functionwhich is inferred from the positioning of basement membranes underneathepithelial layers, and data support the role of collagen IV in tissuestabilization (see, e.g., Cummings et al., supra). Posterior capsuleopacification (PCO) develops as a complication in approximately 20-40%of patients in subsequent years after cataract surgery (see, e.g.,Awasthi et al., Arch Ophthalmol. 2009; 127:555-62). PCO results fromproliferation and activity of residual lens epithelial cells along theposterior capsule in a response akin to wound healing (see, e.g.,Awasthi et al., Arch Ophthalmol. 2009; 127:555-62). Growth factors, suchas fibroblast growth factor, transforming growth factor 13, epidermalgrowth factor, hepatocyte growth factor, insulin-like growth factor, andinterleukins IL-1 and IL-6 may also promote epithelial cell migration,(see, e.g., Awasthi et al., supra; Raj et al., supra). As discussedherein, production of these factors and cytokines by senescent cellscontribute to the SASP. In contrast, in vitro studies show that collagenIV promotes adherence of lens epithelial cells (see, e.g., Olivero etal., Invest. Ophthalmol. Vis. Sci. 1993; 34:2825-34). Adhesion of thecollagen IV, fibronectin, and laminin to the intraocular lens inhibitscell migration and may reduce the risk of PCO (see, e.g., Raj et al.,Int. J. Biomed. Sci. 2007; 3:237-50).

Without wishing to be bound by any particular theory, selective killingof senescent cells by the senolytic agents described herein may slow orimpede (delay, inhibit, retard) the disorganization of the type IVcollagen network. Removal of senescence cells and thereby removing theinflammatory effects of SASP may decrease or inhibit epithelial cellmigration and may also delay (suppress) the onset of presbyopia ordecrease or slow the progressive severity of the condition (such as slowthe advancement from mild to moderate or moderate to severe). Thesenolytic agents described herein may also be useful for post-cataractsurgery to reduce the likelihood of occurrence of PCO.

While no direct evidence for the involvement of cellular senescence withthe development of cataracts has been obtained from human studies, BubR1hypomorphic mice develop posterior subcapsular cataracts bilaterallyearly in life, suggesting that senescence may play a role (see, e.g.,Baker et al., Nat. Cell Biol. 10:825-36 (2008)). Cataracts are aclouding of the lens of an eye, causing blurred vision, and if leftuntreated can result in blindness. Surgery is effective and routinelyperformed to remove cataracts. Administration of one or more of thesenolytic agents described herein may result in decreasing thelikelihood of occurrence of a cataract or may slow or inhibitprogression of a cataract. The presence and severity of a cataract canbe monitored by eye exams using methods routinely performed by a personskilled in the ophthalmology art.

In certain embodiments, at least one senolytic agent that selectivelykills senescent cells may be administered to a subject who is at risk ofdeveloping presbyopia, cataracts, or macular degeneration. Treatmentwith a senolytic agent may be initiated when a human subject is at least40 years of age to delay or inhibit onset or development of cataracts,presbyopia, and macular degeneration. Because almost all humans developpresbyopia, in certain embodiments, the senolytic agent may beadministered in a manner as described herein to a human subject afterthe subject reaches the age of 40 to delay or inhibit onset ordevelopment of presbyopia.

In certain embodiments, the senescence associated disease or disorder isglaucoma. Glaucoma is a broad term used to describe a group of diseasesthat causes visual field loss, often without any other prevailingsymptoms. The lack of symptoms often leads to a delayed diagnosis ofglaucoma until the terminal stages of the disease. Even if subjectsafflicted with glaucoma do not become blind, their vision is oftenseverely impaired. Normally, clear fluid flows into and out of the frontpart of the eye, known as the anterior chamber. In individuals who haveopen/wide-angle glaucoma, this fluid drains too slowly, leading toincreased pressure within the eye. If left untreated, this high pressuresubsequently damages the optic nerve and can lead to complete blindness.The loss of peripheral vision is caused by the death of ganglion cellsin the retina. Ganglion cells are a specific type of projection neuronthat connects the eye to the brain. When the cellular network requiredfor the outflow of fluid was subjected to SA-β-Gal staining, a fourfoldincrease in senescence has been observed in glaucoma patients (see,e.g., Liton et al., Exp. Gerontol. 40:745-748 (2005)).

For monitoring the effect of a therapy on inhibiting progression ofglaucoma, standard automated perimetry (visual field test) is the mostwidely used technique. In addition, several algorithms for progressiondetection have been developed (see, e.g., Wesselink et al., ArchOphthalmol. 127(3):270-274 (2009), and references therein). Additionalmethods include gonioscopy (examines the trabecular meshwork and theangle where fluid drains out of the eye); imaging technology, forexample scanning laser tomography (e.g., HRT3), laser polarimetry (e.g.,GDX), and ocular coherence tomography); ophthalmoscopy; and pachymetermeasurements that determine central corneal thickness.

Metabolic Disease or Disorder.

Senescence-associated diseases or disorders treatable by administering asenolytic agent include metabolic diseases or disorders. Such senescentcell associated diseases and disorders include diabetes, metabolicsyndrome, diabetic ulcers, and obesity.

Diabetes is characterized by high levels of blood glucose caused bydefects in insulin production, insulin action, or both. The greatmajority (90 to 95%) of all diagnosed cases of diabetes in adults aretype 2 diabetes, characterized by the gradual loss of insulin productionby the pancreas. Diabetes is the leading cause of kidney failure,nontraumatic lower-limb amputations, and new cases of blindness amongadults in the U.S. Diabetes is a major cause of heart disease and strokeand is the seventh leading cause of death in the U.S. (see, e.g.,Centers for Disease Control and Prevention, National diabetes factsheet: national estimates and general information on diabetes andpre-diabetes in the United States, 2011 (“Diabetes fact sheet”)).Senolytic agents described herein may be used for treating type 2diabetes, particularly age-, diet- and obesity-associated type 2diabetes.

Involvement of senescent cells in metabolic disease, such as obesity andtype 2 diabetes, has been suggested as a response to injury or metabolicdysfunction (see, e.g., Tchkonia et al., Aging Cell 9:667-684 (2010)).Fat tissue from obese mice showed induction of the senescence markersSA-β-Gal, p53, and p21 (see, e.g., Tchkonia et al., supra; Minamino etal., Nat. Med. 15:1082-1087 (2009)). A concomitant up-regulation ofpro-inflammatory cytokines, such as tumor necrosis factor-α andCcl2/MCP1, was observed in the same fat tissue (see, e.g., Minamino etal., supra). Induction of senescent cells in obesity potentially hasclinical implications because pro-inflammatory SASP components are alsosuggested to contribute to type 2 diabetes (see, e.g., Tchkonia et al.,supra). A similar pattern of up-regulation of senescence markers andSASP components are associated with diabetes, both in mice and in humans(see, e.g., Minamino et al., supra). Accordingly, the methods describedherein that comprise administering a senolytic agent may be useful fortreatment or prophylaxis of type 2 diabetes, as well as obesity andmetabolic syndrome. Without wishing to be bound by theory, contact ofsenescent pre-adipocytes with a senolytic agent thereby killing thesenescent pre-adipocytes may provide clinical and health benefit to aperson who has any one of diabetes, obesity, or metabolic syndrome.

Subjects suffering from type 2 diabetes can be identified using standarddiagnostic methods known in the art for type 2 diabetes. Generally,diagnosis of type 2 diabetes is based on symptoms (e.g., increasedthirst and frequent urination, increased hunger, weight loss, fatigue,blurred vision, slow-healing sores or frequent infections, and/or areasof darkened skin), medical history, and/or physical examination of apatient. Subjects at risk of developing type 2 diabetes include thosewho have a family history of type 2 diabetes and those who have otherrisk factors such as excess weight, fat distribution, inactivity, race,age, prediabetes, and/or gestational diabetes.

The effectiveness of a senolytic agent can readily be determined by aperson skilled in the medical and clinical arts. One or any combinationof diagnostic methods, including physical examination, assessment andmonitoring of clinical symptoms, and performance of analytical tests andmethods, such as those described herein, may be used for monitoring thehealth status of the subject. A subject who is receiving one or moresenolytic agents described herein for treatment or prophylaxis ofdiabetes can be monitored, for example, by assaying glucose and insulintolerance, energy expenditure, body composition, fat tissue, skeletalmuscle, and liver inflammation, and/or lipotoxicity (muscle and liverlipid by imaging in vivo and muscle, liver, bone marrow, and pancreaticβ-cell lipid accumulation and inflammation by histology). Othercharacteristic features or phenotypes of type 2 diabetes are known andcan be assayed as described herein and by using other methods andtechniques known and routinely practiced in the art.

Obesity and obesity-related disorders are used to refer to conditions ofsubjects who have a body mass that is measurably greater than ideal fortheir height and frame. Body Mass index (BMi) is a measurement tool usedto determine excess body weight, and is calculated from the height andweight of a subject. A human is considered overweight when the personhas a BMI of 25-29; a person is considered obese when the person has aBMI of 30-39, and a person is considered severely obese when the personhas a BMI of ≥40. Accordingly, the terms obesity and obesity-relatedrefer to human subjects with body mass index values of greater than 30,greater than 35, or greater than 40. A category of obesity not capturedby BMI is called “abdominal obesity” in the art, which relates to theextra fat found around a subject's middle, which is an important factorin health, even independent of BMI. The simplest and most often usedmeasure of abdominal obesity is waist size. Generally abdominal obesityin women is defined as a waist size 35 inches or higher, and in men as awaist size of 40 inches or higher. More complex methods for determiningobesity require specialized equipment, such as magnetic resonanceimaging or dual energy X-ray absorptioimetry machines.

A condition or disorder associated with diabetes and senescence is adiabetic ulcer (i.e., diabetic wound). An ulcer is a breakdown in theskin, which may extend to involve the subcutaneous tissue or even muscleor bone. These lesions occur, particularly, on the lower extremities.Patients with diabetic venous ulcer exhibit elevated presence ofcellular senescence at sites of chronic wounds (see, e.g., Stanley etal. (2001) J. Vas. Surg. 33: 1206-1211). Chronic inflammation is alsoobserved at sites of chronic wounds, such as diabetic ulcers (see, e.g.,Goren et al. (2006) Am. J. Pathol. 7 168: 65-77; Seitz et al. (2010)Exp. Diabetes Res. 2010: 476969), suggesting that the proinflammatorycytokine phenotype of senescent cells has a role in the pathology.

Subjects who have type 2 diabetes or who are at risk of developing type2 diabetes may have metabolic syndrome. Metabolic syndrome in humans istypically associated with obesity and characterized by one or more ofcardiovascular disease, liver steatosis, hyperlipidemia, diabetes, andinsulin resistance. A subject with metabolic syndrome may present with acluster of metabolic disorders or abnormalities which may include, forexample, one or more of hypertension, type-2 diabetes, hyperlipidemia,dyslipidemia (e.g., hypertriglyceridemia, hypercholesterolemia), insulinresistance, liver steatosis (steatohepatitis), hypertension,atherosclerosis, and other metabolic disorders.

Renal Dysfunction:

Nephrological pathologies, such as glomerular disease, arise in theelderly. Glomerulonephritis is characterized by inflammation of thekidney and by the expression of two proteins, IL1α and IL1β (see, e.g.,Niemir et al. (1997) Kidney Int. 52:393-403). IL1α and IL1β areconsidered master regulators of SASP (see, e.g., Coppe et al. (2008)PLoS. Biol. 6: 2853-68). Glomerular disease is associated with elevatedpresence of senescent cells, especially in fibrotic kidneys (see, e.g.,Sis et al. (2007) Kidney Int. 71:218-226).

Dermatological Disease or Disorder.

Senescence-associated diseases or disorders treatable by administering asenolytic agent described herein include dermatological diseases ordisorders. Such senescent cell associated diseases and disorders includepsoriasis and eczema, which are also inflammatory diseases and arediscussed in greater detail above. Other dermatological diseases anddisorders that are associated with senescence include rhytides (wrinklesdue to aging); pruritis (linked to diabetes and aging); dysesthesia(chemotherapy side effect that is linked to diabetes and multiplesclerosis); psoriasis (as noted) and other papulosquamous disorders, forexample, erythroderma, lichen planus, and lichenoid dermatosis; atopicdermatitis (a form of eczema and associated with inflammation);eczematous eruptions (often observed in aging patients and linked toside effects of certain drugs). Other dermatological diseases anddisorders associated with senescence include eosinophilic dermatosis(linked to certain kinds of hemotologic cancers); reactive neutrophilicdermatosis (associated with underlying diseases such as inflammatorybowel syndrome); pemphigus (an autoimmune disease in whichautoantibodies form against desmoglein); pemphigoid and otherimmunobullous dermatosis (autoimmune blistering of skin);fibrohistocytic proliferations of skin, which is linked to aging; andcutaneous lymphomas that are more common in older populations. Anotherdermatological disease that may be treatable according to the methodsdescribed herein includes cutaneous lupus, which is a symptom of lupuserythematosus. Late onset lupus may be linked to decreased (i.e.,reduced) function of T-cell and B-cells and cytokines (immunosenescence)associated with aging.

Metastasis.

In a particular embodiment, methods are provided for treating orpreventing (i.e., reducing the likelihood of occurrence or developmentof) a senescence cell associated disease (or disorder or condition),which is metastasis. The senolytic agents described herein may also beused according to the methods described herein for treating orpreventing (i.e., reducing the likelihood of occurrence of) metastasis(i.e., the spreading and dissemination of cancer or tumor cells) fromone organ or tissue to another organ or tissue in the body.

A senescent cell-associated disease or disorder includes metastasis, anda subject who has a cancer may benefit from administration of asenolytic agent as described herein for inhibiting metastasis. Such asenolytic agent when administered to a subject who has a canceraccording to the methods described herein may inhibit tumorproliferation. Metastasis of a cancer occurs when the cancer cells(i.e., tumor cells) spread beyond the anatomical site of origin andinitial colonization to other areas throughout the body of the subject.Tumor proliferation may be determined by tumor size, which can bemeasured in various ways familiar to a person skilled in the art, suchas by PET scanning, MRI, CAT scan, biopsy, for example. The effect ofthe therapeutic agent on tumor proliferation may also be evaluated byexamining differentiation of the tumor cells.

As used herein and in the art, the terms cancer or tumor are clinicallydescriptive terms that encompass diseases typically characterized bycells exhibiting abnormal cellular proliferation. The term cancer isgenerally used to describe a malignant tumor or the disease statearising from the tumor. Alternatively, an abnormal growth may bereferred to in the art as a neoplasm. The term tumor, such as inreference to a tissue, generally refers to any abnormal tissue growththat is characterized, at least in part, by excessive and abnormalcellular proliferation. A tumor may be metastatic and capable ofspreading beyond its anatomical site of origin and initial colonizationto other areas throughout the body of the subject. A cancer may comprisea solid tumor or may comprise a “liquid” tumor (e.g., leukemia and otherblood cancers).

Cells are induced to senesce by cancer therapies, such as radiation andcertain chemotherapy drugs. The presence of senescent cells increasessecretion of inflammatory molecules (see description herein of senescentcells), promotes tumor progression, which may include promoting tumorgrowth and increasing tumor size, promoting metastasis, and alteringdifferentiation. When senescent cells are destroyed, tumor progressionis significantly inhibited, resulting in tumors of small size and withlittle or no observed metastatic growth (see, e.g., Int'l Appl.Publication No. WO 2013/090645).

In one embodiment, methods are provided for preventing (i.e., reducingthe likelihood of occurrence of), inhibiting, or retarding metastasis ina subject who has a cancer by administering a senolytic agent asdescribed herein. In a particular embodiment, the senolytic agent isadministered on one or more days within a treatment window (i.e.,treatment course) of no longer than 7 days or 14 days. In otherembodiments, the treatment course is no longer than 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or no longer than 21days. In other embodiments, the treatment course is a single day. Incertain embodiments, the senolytic agent is administered on two or moredays within a treatment window of no longer than 7 days or 14 days, on 3or more days within a treatment window of no longer than 7 days or 14days; on 4 or more days within a treatment window of no longer than 7days or 14 days; on 5 or more days within a treatment window of nolonger than 7 days or 14 days; on 6, 7, 8, 9, 10, 11, 12, 13, or 14 dayswithin treatment window of no longer than 7 days or 14 days. In certainembodiments, when the at least one senolytic agent is administered to asubject for a treatment window of 3 days or more, the agent may beadministered every 2^(nd) day (i.e., every other day). In other certainembodiments when the at least one senolytic agent is administered to asubject for a treatment window of 4 days or more, the agent may beadministered every 3^(rd) day (i.e., every other third day).

Because cells may be induced to senesce by cancer therapies, such asradiation and certain chemotherapy drugs (e.g., doxorubicin; paclitaxel;gemcitabine; pomalidomide; lenalidomide), a senolytic agent describedherein may be administered after the chemotherapy or radiotherapy tokill (or facilitate killing) of these senescent cells. As discussedherein and understood in the art, establishment of senescence, such asshown by the presence of a senescence-associated secretory phenotype(SASP), occurs over several days; therefore, administering a senolyticagent to kill senescent cells, and thereby reduce the likelihood ofoccurrence or reduce the extent of metastasis, is initiated whensenescence has been established. As discussed herein, the followingtreatment courses for administration of the senolytic agent may be usedin methods described herein for treating or preventing (i.e., reducingthe likelihood of occurrence, or reducing the severity) a chemotherapyor radiotherapy side effect.

In certain embodiments, when chemotherapy or radiotherapy isadministered in a treatment cycle of at least one day on-therapy (i.e.,chemotherapy or radiotherapy)) followed by at least 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 (or about 2 weeks), 15, 16, 17, 18, 19, 20, 21 (orabout 3 weeks) days, or about 4 weeks (about one month) off-therapy(i.e., off chemo- or radio-therapy), the senolytic agent is administeredon one or more days during the off-therapy time interval (time period)beginning on or after the second day of the off-therapy time intervaland ending on or before the last day of the off-therapy time interval.By way of illustrative example, if n is the number of days off-therapy,then the senolytic agent is administered on at least one day and no morethan n−1 days of the off-therapy time interval. In a certain particularembodiment when chemotherapy or radiotherapy is administered in atreatment cycle of at least one day on-therapy (i.e., chemotherapy orradiotherapy)) followed by at least one week off-therapy, the senolyticagent is administered on one or more days during the off-therapy timeinterval beginning on or after the second day of the off-therapy timeinterval and ending on or before the last day of the off-therapy timeinterval. In a more specific embodiment, when chemotherapy orradiotherapy is administered in a treatment cycle of at least one dayon-therapy (i.e., chemotherapy or radiotherapy)) followed by at leastone week off-therapy, the senolytic agent is administered on one daythat is the sixth day of the off-therapy time interval. In otherspecific embodiments, when chemotherapy or radiotherapy is administeredin a treatment cycle of at least one day on-therapy (i.e., chemotherapyor radiotherapy)) followed by at least two weeks off-therapy, thesenolytic agent is administered beginning on the sixth day of theoff-chemo- or radio-therapy time interval and ending at least one day orat least two days prior to the first day of a subsequent chemotherapy orradiation therapy treatment course. By way of example, if the off-chemo-or radio-therapy time interval is two weeks, a senolytic agent may beadministered on at least one and on no more than 7 days (i.e., 1, 2, 3,4, 5, 6, or 7 days) of the off-therapy time interval beginning on thesixth day after the chemotherapy or radiotherapy course ends (i.e., thesixth day of the off chemo-radio-therapy interval). When the off-chemo-or radio-therapy time interval is at least three weeks, a senolyticagent may be administered on at least one day and on no more than 14days (i.e., 1-14 days: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14days) of the off-therapy time interval beginning on the sixth day afterthe chemotherapy or radiotherapy course ends. In other embodiments,depending on the off-chemo-radio-therapy interval, the senolytic agenttreatment course is at least one day and no longer than 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or no more than 21days (i.e., 1-21 days), provided that administration of the senolyticagent is not concurrent with the chemotherapy or radiotherapy. Incertain embodiments, the senolytic agent treatment course is a singleday. In certain embodiments, the senolytic agent is administered on twoor more days within a treatment window of no longer than 14 days, on 3or more days within a treatment window of no longer than 14 days; on 4or more days within a treatment window of no longer than 14 days; on 5or more days within a treatment window of no longer than 14 days; on 6,7, 8, 9, 10, 11, 12, 13, or 14 days within treatment window of no longerthan 14 days. In certain embodiments, when the at least one senolyticagent is administered to a subject during a treatment course of 3 daysor more, the agent may be administered every 2^(nd) day (i.e., everyother day). In other certain embodiments when the at least one senolyticagent is administered to a subject during a treatment course of 4 daysor more, the agent may be administered every 3^(rd) day (i.e., everyother third day).

Many chemotherapy and radiotherapy treatment regimens comprise a finitenumber of cycles of on-drug therapy followed by off-drug therapy orcomprise a finite timeframe in which the chemotherapy or radiotherapy isadministered. Such cancer treatment regimens may also be calledtreatment protocols. The protocols are determined by clinical trials,drug labels, and clinical staff in conjunction with the subject to betreated. The number of cycles of a chemotherapy or radiotherapy or thetotal length of time of a chemotherapy or radiotherapy regimen can varydepending on the patient's response to the cancer therapy. The timeframefor such treatment regimens is readily determined by a person skilled inthe oncology art. In another embodiment for treating metastasis, asenolytic agent may be administered after the treatment regimen ofchemotherapy or radiotherapy has been completed. In a particularembodiment, the senolytic agent is administered after the chemotherapyor radiotherapy has been completed on one or more days within treatmentwindow (i.e., senolytic agent treatment course) of no longer than 14days. In other embodiments, the senolytic agent treatment course is nolonger than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or no more than 21 days. In other embodiments, the treatmentcourse is a single day. In certain embodiments, the senolytic agent isadministered on two or more days within a treatment window of no longerthan 14 days, on 3 or more days within a treatment window of no longerthan 14 days; on 4 or more days within a treatment window of no longerthan 14 days; on 5 or more days within a treatment window of no longerthan 14 days; on 6, 7, 8, 9, 10, 11, 12, 13, or 14 days within treatmentwindow of no longer than 14 days. In certain embodiments, when the atleast one senolytic agent is administered to a subject afterchemotherapy or radiotherapy for a treatment window of 3 days or more,the agent may be administered every 2^(nd) day (i.e., every other day).In other certain embodiments when the at least one senolytic agent isadministered to a subject for a treatment window of 4 days or more, theagent may be administered every 3^(rd) d day (i.e., every other thirdday). In one embodiment, the treatment with the senolytic agent may beinitiated at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 daysor later after the cancer treatment regimen has been completed. In amore particular embodiment, the treatment with the senolytic agent maybe initiated at least 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or laterafter the cancer treatment regimen has been completed. Any of theadditional treatment courses and treatment cycles for administration ofa senolytic agent described herein may be followed for inhibitingmetastasis in a subject after a chemotherapy or radiotherapy protocolhas been completed.

A chemotherapy may be referred to as a chemotherapy, chemotherapeutic,or chemotherapeutic drug. Many chemotherapeutics are compounds referredto as small organic molecules. Chemotherapy is a term that is also usedto describe a combination chemotherapeutic drugs that are administeredto treat a particular cancer. As understood by a person skilled in theart, a chemotherapy may also refer to a combination of two or morechemotherapeutic molecules that are administered coordinately and whichmay be referred to as combination chemotherapy. Numerouschemotherapeutic drugs are used in the oncology art and include, withoutlimitation, alkylating agents; antimetabolites; anthracyclines, plantalkaloids; and topoisomerase inhibitors.

A cancer that may metastasize may be a solid tumor or may be a liquidtumor (e.g., a blood cancer, for example, a leukemia). Cancers that areliquid tumors are classified in the art as those that occur in blood,bone marrow, and lymph nodes and include generally, leukemias (myeloidand lymphocytic), lymphomas (e.g., Hodgkin lymphoma), and melanoma(including multiple myeloma). Leukemias include for example, acutelymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), andhairy cell leukemia. Cancers that are solid tumors and occur in greaterfrequency in humans include, for example, prostate cancer, testicularcancer, breast cancer, brain cancer, pancreatic cancer, colon cancer,thyroid cancer, stomach cancer, lung cancer, ovarian cancer, Kaposi'ssarcoma, skin cancer (including squamous cell skin cancer), renalcancer, head and neck cancers, throat cancer, squamous carcinomas thatform on the moist mucosal linings of the nose, mouth, throat, etc.),bladder cancer, osteosarcoma (bone cancer), cervical cancer, endometrialcancer, esophageal cancer, liver cancer, and kidney cancer. In certainspecific embodiments, the senescent cell-associated disease or disordertreated or prevented (i.e., likelihood of occurrence or development isreduced) by the methods described herein is metastasis of melanomacells, prostate cancer cells, testicular cancer cells, breast cancercells, brain cancer cells, pancreatic cancer cells, colon cancer cells,thyroid cancer cells, stomach cancer cells, lung cancer cells, ovariancancer cells, Kaposi's sarcoma cells, skin cancer cells, renal cancercells, head or neck cancer cells, throat cancer cells, squamouscarcinoma cells, bladder cancer cells, osteosarcoma cells, cervicalcancer cells, endometrial cancer cells, esophageal cancer cells, livercancer cells, or kidney cancer cells.

The methods described herein are also useful for inhibiting, retardingor slowing progression of metastatic cancer of any one of the types oftumors described in the medical art. Types of cancers (tumors) includethe following: adrenocortical carcinoma, childhood adrenocorticalcarcinoma, aids-related cancers, anal cancer, appendix cancer, basalcell carcinoma, childhood basal cell carcinoma, bladder cancer,childhood bladder cancer, bone cancer, brain tumor, childhoodastrocytomas, childhood brain stem glioma, childhood central nervoussystem atypical teratoid/rhabdoid tumor, childhood central nervoussystem embryonal tumors, childhood central nervous system germ celltumors, childhood craniopharyngioma brain tumor, childhood ependymomabrain tumor, breast cancer, childhood bronchial tumors, carcinoid tumor,childhood carcinoid tumor, gastrointestinal carcinoid tumor, carcinomaof unknown primary, childhood carcinoma of unknown primary, childhoodcardiac (heart) tumors, cervical cancer, childhood cervical cancer,childhood chordoma, chronic myeloproliferative disorders, colon cancer,colorectal cancer, childhood colorectal cancer, extrahepatic bile ductcancer, ductal carcinoma in situ (DCIS), endometrial cancer, esophagealcancer, childhood esophageal cancer, childhood esthesioneuroblastoma,eye cancer, malignant fibrous histiocytoma of bone, gallbladder cancer,gastric (stomach) cancer, childhood gastric (stomach) cancer,gastrointestinal stromal tumors (GIST), childhood gastrointestinalstromal tumors (GIST), childhood extracranial germ cell tumor,extragonadal germ cell tumor, gestational trophoblastic tumor, glioma,head and neck cancer, childhood head and neck cancer, hepatocellular(liver) cancer, hypopharyngeal cancer, kidney cancer, renal cell kidneycancer, Wilms tumor, childhood kidney tumors, Langerhans cellhistiocytosis, laryngeal cancer, childhood laryngeal cancer, leukemia,acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (cml),hairy cell leukemia, lip cancer, liver cancer (primary), childhood livercancer (primary), lobular carcinoma in situ (LCIS), lung cancer,non-small cell lung cancer, small cell lung cancer, lymphoma,aids-related lymphoma, burkitt lymphoma, cutaneous t-cell lymphoma,Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous systemlymphoma (CNS), melanoma, childhood melanoma, intraocular (eye)melanoma, Merkel cell carcinoma, malignant mesothelioma, childhoodmalignant mesothelioma, metastatic squamous neck cancer with occultprimary, midline tract carcinoma involving NUTgene, mouth cancer,childhood multiple endocrine neoplasia syndromes, mycosis fungoides,myelodysplastic syndromes, myelodysplastic neoplasms, myeloproliferativeneoplasms, multiple myeloma, nasal cavity cancer, nasopharyngeal cancer,childhood nasopharyngeal cancer, neuroblastoma, oral cancer, childhoodoral cancer, oropharyngeal cancer, ovarian cancer, childhood ovariancancer, epithelial ovarian cancer, low malignant potential tumor ovariancancer, pancreatic cancer, childhood pancreatic cancer, pancreaticneuroendocrine tumors (islet cell tumors), childhood papillomatosis,paraganglioma, paranasal sinus cancer, parathyroid cancer, penilecancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasmacell neoplasm, childhood pleuropulmonary blastoma, prostate cancer,rectal cancer, renal pelvis transitional cell cancer, retinoblastoma,salivary gland cancer, childhood salivary gland cancer, Ewing sarcomafamily of tumors, Kaposi Sarcoma, osteosarcoma, rhabdomyosarcoma,childhood rhabdomyosarcoma, soft tissue sarcoma, uterine sarcoma, Sézarysyndrome, childhood skin cancer, nonmelanoma skin cancer, smallintestine cancer, squamous cell carcinoma, childhood squamous cellcarcinoma, testicular cancer, childhood testicular cancer, throatcancer, thymoma and thymic carcinoma, childhood thymoma and thymiccarcinoma, thyroid cancer, childhood thyroid cancer, ureter transitionalcell cancer, urethral cancer, endometrial uterine cancer, vaginalcancer, vulvar cancer, Waldenström macroglobulinemia.

Chemotherapy and Radiotherapy Side Effects.

In another embodiment, the senescence cell associated disorder orcondition is a chemotherapeutic side effect or a radiotherapy sideeffect. Examples of chemotherapeutic agents that include non-cancercells to senesce include anthracyclines (such as doxorubicin,daunorubicin); taxols (e.g., paclitaxel); gemcitabine; pomalidomide; andlenalidomide. One or more of the senolytic agents administered asdescribed herein may be used for treating and/or preventing (i.e.,reducing the likelihood of occurrence of) a chemotherapeutic side effector a radiotherapy side effect. Removal or destruction of senescent cellsmay ameliorate acute toxicity, including acute toxicity comprisingenergy imbalance, of a chemotherapy or radiotherapy. Acute toxic sideeffects include but are not limited to gastrointestinal toxicity (e.g.,nausea, vomiting, constipation, anorexia, diarrhea), peripheralneuropathy, fatigue, malaise, low physical activity, hematologicaltoxicity (e.g., anemia), hepatotoxicity, alopecia (hair loss), pain,infection, mucositis, fluid retention, dermatological toxicity (e.g.,rashes, dermatitis, hyperpigmentation, urticaria, photosensitivity, nailchanges), mouth (e.g., oral mucositis), gum or throat problems, or anytoxic side effect caused by a chemotherapy or radiotherapy. For example,toxic side effects caused by radiotherapy or chemotherapy (see, e.g.,National Cancer Institute web site) may be ameliorated by the methodsdescribed herein. Accordingly, in certain embodiments, methods areprovided herein for ameliorating (reducing, inhibiting, or preventingoccurrence (i.e., reducing the likelihood of occurrence)) acute toxicityor reducing severity of a toxic side effect (i.e., deleterious sideeffect) of a chemotherapy or radiotherapy or both in a subject whoreceives the therapy, wherein the method comprises administering to thesubject an agent that selectively kills, removes, or destroys orfacilitates selective destruction of senescent cells. Administration ofa senolytic agent for treating or reducing the likelihood of occurrence,or reducing the severity of a chemotherapy or radiotherapy side effectmay be accomplished by the same treatment courses described above fortreatment/prevention of metastasis. As described for treating orpreventing (i.e., reducing the likelihood of occurrence of) metastasis,the senolytic agent is administered during the off-chemotherapy oroff-radiotherapy time interval or after the chemotherapy or radiotherapytreatment regimen has been completed.

In a more specific embodiment, the acute toxicity is an acute toxicitycomprising energy imbalance and may comprise one or more of weight loss,endocrine change(s) (e.g., hormone imbalance, change in hormonesignaling), and change(s) in body composition. In certain embodiments,an acute toxicity comprising energy imbalance relates to decreased orreduced ability of the subject to be physically active, as indicated bydecreased or diminished expenditure of energy than would be observed ina subject who did not receive the medical therapy. By way ofnon-limiting example, such an acute toxic effect that comprises energyimbalance includes low physical activity. In other particularembodiments, energy imbalance comprises fatigue or malaise.

In one embodiment, a chemotherapy side effect to be treated or prevented(i.e., likelihood of occurrence is reduced) by a senolytic agent iscardiotoxicity. A subject who has a cancer that is being treated with ananthracycline (such as doxorubicin, daunorubicin) may be treated withone or more senolytic agents described herein that reduce, ameliorate,or decrease the cardiotoxicity of the anthracycline. As is wellunderstood in the medical art, because of the cardiotoxicity associatedwith anthracyclines, the maximum lifetime dose that a subject canreceive is limited even if the cancer is responsive to the drug.Administration of one or more of the senolytic agents may reduce thecardiotoxicity such that additional amounts of the anthracycline can beadministered to the subject, resulting in an improved prognosis relatedto cancer disease. In one embodiment, the cardiotoxicity results fromadministration of an anthracyline, such as doxorubicin. Doxorubicin isan anthracycline topoisomerase that is approved for treating patientswho have ovarian cancer after failure of a platinum based therapy;Kaposi's sarcoma after failure of primary systemic chemotherapy orintolerance to the therapy; or multiple myeloma in combination withbortezomib in patients who have not previously received bortezomib orwho have received at least one prior therapy. Doxorubicin may causemyocardial damage that could lead to congestive heart failure if thetotal lifetime dose to a patient exceeds 550 mg/m². Cardiotoxicity mayoccur at even lower doses if the patient also receives mediastinalirradiation or another cardiotoxic drug. See drug product inserts (e.g.,DOXIL, ADRIAMYCIN).

In other embodiments, a senolytic agent described herein may be used inthe methods as provided herein for ameliorating chronic or long termside effects. Chronic toxic side effects typically result from multipleexposures to or administrations of a chemotherapy or radiotherapy over alonger period of time. Certain toxic effects appear long after treatment(also called late toxic effects) and result from damage to an organ orsystem by the therapy. Organ dysfunction (e.g., neurological, pulmonary,cardiovascular, and endocrine dysfunction) has been observed in patientswho were treated for cancers during childhood (see, e.g., Hudson et al.,JAMA 309:2371-81 (2013)). Without wishing to be bound by any particulartheory, by destroying senescent cells, particular normal cells that havebeen induced to senescence by chemotherapy or radiotherapy, thelikelihood of occurrence of a chronic side effect may be reduced, or theseverity of a chronic side effect may be reduced or diminished, or thetime of onset of a chronic side effect may be delayed. Chronic and/orlate toxic side effects that occur in subjects who received chemotherapyor radiation therapy include by way of non-limiting example,cardiomyopathy, congestive heart disease, inflammation, early menopause,osteoporosis, infertility, impaired cognitive function, peripheralneuropathy, secondary cancers, cataracts and other vision problems,hearing loss, chronic fatigue, reduced lung capacity, and lung disease.

In addition, by killing or removing senescent cells in a subject who hasa cancer by administering a senolytic agent, the sensitivity to thechemotherapy or the radiotherapy may be enhanced in a clinically orstatistically significant manner than if the senolytic agent was notadministered. In other words, development of chemotherapy orradiotherapy resistance may be inhibited when a senolytic agent isadministered to a subject treated with the respective chemotherapy orradiotherapy.

Age-Related Diseases and Disorders.

A senolytic agent may also be useful for treating or preventing (i.e.,reducing the likelihood of occurrence) of an age-related disease ordisorder that occurs as part of the natural aging process or that occurswhen the subject is exposed to a senescence inducing agent or factor(e.g., irradiation, chemotherapy, smoking tobacco, high-fat/high sugardiet, other environmental factors). An age-related disorder or diseaseor an age-sensitive trait may be associated with a senescence-inducingstimulus. The efficacy of a method of treatment described herein may bemanifested by reducing the number of symptoms of an age-related disorderor age-sensitive trait associated with a senescence-inducing stimulus,decreasing the severity of one or more symptoms, or delaying theprogression of an age-related disorder or age-sensitive trait associatedwith a senescence-inducing stimulus. In other particular embodiments,preventing an age-related disorder or age-sensitive trait associatedwith a senescence-inducing stimulus refers to preventing (i.e., reducingthe likelihood of occurrence) or delaying onset of an age-relateddisorder or age-sensitive trait associated with a senescence-inducingstimulus, or reoccurrence of one or more age-related disorder orage-sensitive trait associated with a senescence-inducing stimulus. Agerelated diseases or conditions include, for example, renal dysfunction,kyphosis, herniated intervertebral disc, frailty, hair loss, hearingloss, vision loss (blindness or impaired vision), muscle fatigue, skinconditions, skin nevi, diabetes, metabolic syndrome, and sarcopenia.Vision loss refers to the absence of vision when a subject previouslyhad vision. Various scales have been developed to describe the extent ofvision and vision loss based on visual acuity. Age-related diseases andconditions also include dermatological conditions, for example withoutlimitation, treating one or more of the following conditions: wrinkles,including superficial fine wrinkles; hyperpigmentation; scars; keloid;dermatitis; psoriasis; eczema (including seborrheic eczema); rosacea;vitiligo; ichthyosis vulgaris; dermatomyositis; and actinic keratosis.

Frailty has been defined as a clinically recognizable state of increasedvulnerability resulting from aging-associated decline in reserve andfunction across multiple physiologic systems that compromise a subject'sability to cope with every day or acute stressors. Frailty has been maybe characterized by compromised energetics characteristics such as lowgrip strength, low energy, slowed waking speed, low physical activity,and/or unintentional weight loss. Studies have suggested that a patientmay be diagnosed with frailty when three of five of the foregoingcharacteristics are observed (see, e.g., Fried et al., J. Gerontol. ABiol. Sci. Med, Sci. 2001; 56(3):M146-M156; Xue, Clin. Geriatr. Med.2011; 27(1):1-15). In certain embodiments, aging and diseases anddisorders related to aging may be treated or prevented (i.e., thelikelihood of occurrence of is reduced) by administering a senolyticagent. The senolytic agent may inhibit senescence of adult stem cells orinhibit accumulation, kill, or facilitate removal of adult stem cellsthat have become senescent. See, e.g., Park et al., J. Clin. Invest.113:175-79 (2004) and Sousa-Victor, Nature 506:316-21 (2014) describingimportance of preventing senescence in stem cells to maintainregenerative capacity of tissues.

The effectiveness of a senolytic agent with respect to treating asenescence-associated disease or disorder described herein can readilybe determined by a person skilled in the medical and clinical arts. Oneor any combination of diagnostic methods appropriate for the particulardisease or disorder, which methods are well known to a person skilled inthe art, including physical examination, patient self-assessment,assessment and monitoring of clinical symptoms, performance ofanalytical tests and methods, including clinical laboratory tests,physical tests, and exploratory surgery, for example, may be used formonitoring the health status of the subject and the effectiveness of thesenolytic agent. The effects of the methods of treatment describedherein can be analyzed using techniques known in the art, such ascomparing symptoms of patients suffering from or at risk of a particulardisease or disorder that have received the pharmaceutical compositioncomprising a senolytic agent with those of patients who were not treatedwith the senolytic agent or who received a placebo treatment.

As understood by a person skilled in the medical art, the terms, “treat”and “treatment,” refer to medical management of a disease, disorder, orcondition of a subject (i.e., patient) (see, e.g., Stedman's MedicalDictionary). In general, an appropriate dose and treatment regimenprovide the senolytic agent in an amount sufficient to providetherapeutic and/or prophylactic benefit. Therapeutic benefit forsubjects to whom the senolytic agents described herein are administered,includes, for example, an improved clinical outcome, wherein the objectis to prevent or slow or retard (lessen) an undesired physiologicalchange associated with the disease, or to prevent or slow or retard(lessen) the expansion or severity of such disease. As discussed herein,effectiveness of the one or more senolytic agents may include beneficialor desired clinical results that comprise, but are not limited to,abatement, lessening, or alleviation of symptoms that result from or areassociated with the disease to be treated; decreased occurrence ofsymptoms; improved quality of life; longer disease-free status (i.e.,decreasing the likelihood or the propensity that a subject will presentsymptoms on the basis of which a diagnosis of a disease is made);diminishment of extent of disease; stabilized (i.e., not worsening)state of disease; delay or slowing of disease progression; ameliorationor palliation of the disease state; and remission (whether partial ortotal), whether detectable or undetectable; and/or overall survival. Theeffectiveness of the senolytic agents described herein may also meanprolonging survival when compared to expected survival if a subject werenot receiving the senolytic agent that selectively kills senescentcells.

Administration of a senolytic agent described herein can prolongprolonging survival when compared to expected survival if a subject werenot receiving treatment. Subjects in need of treatment include those whoalready have the disease or disorder as well as subjects prone to haveor at risk of developing the disease or disorder, and those in which thedisease, condition, or disorder is to be treated prophylactically. Asubject may have a genetic predisposition for developing a disease ordisorder that would benefit from clearance of senescent cells or may beof a certain age wherein receiving a senolytic agent would provideclinical benefit to delay development or reduce severity of a disease,including an age-related disease or disorder.

In another embodiment, a method is provided for treating asenescence-associated disease or disorder that further comprisesidentifying a subject who would benefit from treatment with a senolyticagent described herein (i.e., phenotyping; individualized treatment).This method comprises first detecting the level of senescent cells inthe subject, such as in a particular organ or tissue of the subject. Abiological sample may be obtained from the subject, for example, a bloodsample, serum or plasma sample, biopsy specimen, body fluids (e.g., lunglavage, ascites, mucosal washings, synovial fluid, vitreous fluid,spinal fluid), bone marrow, lymph nodes, tissue explant, organ culture,or any other tissue or cell preparation from a subject. The level ofsenescent cells may be determined according to any of the in vitroassays or techniques described herein. For example, senescence cells maybe detected by morphology (as viewed by microscopy, for example);production of senescence associated markers such as,senescence-associated β-galactosidase (SA-β-gal), p161NK4a, p21, PAI-1,or any one or more SASP factors (e.g., IL-6, MMP3). The senescent cellsand non-senescent cells of the biological sample may also be used in anin vitro cell assay in which the cells are exposed to any one of thesenolytic agents described herein to determine the capability of thesenolytic agent to kill the subject's senescent cells without undesiredtoxicity to non-senescent cells. As positive controls in these assays,the assay may incorporate any one of the senolytic agents (e.g.,Nutlin-3a, RG-7112, ABT-263, ABT-737, WEHI-539, A-1155463, MK-2206)described herein. The subject then may be treated with an appropriatesenolytic agent, which may be a MDM2 inhibitor; an inhibitor of one ormore Bcl-2 anti-apoptotic protein family members wherein the inhibitorinhibits at least Bcl-xL (e.g., a Bcl-xL selective inhibitor,Bcl-2/Bcl-xL/Bcl-w inhibitor, a Bcl-2/Bcl-xL or a Bcl-xL/Bcl-winhibitor); or an Akt specific inhibitor. In addition, these methods maybe used to monitor the level of senescent cells in the subject before,during, and after treatment with a senolytic agent. In certainembodiments, the presence of senescence cells, may be detected (e.g., bydetermining the level of a senescent cell marker expression of mRNA, forexample), and the treatment course and/or non-treatment interval can beadjusted accordingly.

A subject, patient, or individual in need of treatment with a senolyticagent as described herein may be a human or may be a non-human primateor other animal (i.e., veterinary use) who has developed symptoms of asenescence cell-associated disease or disorder or who is at risk fordeveloping a senescence cell-associated disease or disorder. Non-humananimals that may be treated include mammals, for example, non-humanprimates (e.g., monkey, chimpanzee, gorilla, and the like), rodents(e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs,swine (e.g., pig, miniature pig), equine, canine, feline, bovine,elephants, bears and other domestic, farm, and zoo animals.

Methods for Characterizing and Identifying Senolytic AgentsCharacterizing a senolytic agent can be determined using one or morecell-based assays and one or more animal models described herein or inthe art and with which a person skilled in the art will be familiar. Asenolytic agent is an agent that selectively kills or destroys asenescent cell in a statistically significant, clinically significant,or biologically significant manner. A senolytic agent may selectivelykill one or more types of senescent cells (e.g., senescentpreadipocytes, senescent endothelial cells, senescent fibroblasts,senescent neurons, senescent epithelial cells, senescent mesenchymalcells, senescent smooth muscle cells, senescent macrophages, orsenescent chondrocytes). In certain particular embodiments, a senolyticagent is capable of selectively killing at least senescent fibroblasts.

Characterizing an agent as a senolytic agent can be accomplished usingone or more cell-based assays and one or more animal models describedherein or in the art. A person skilled in the art will readilyappreciate that characterizing an agent as a senolytic agent anddetermining the level of killing by an agent can be accomplished bycomparing the activity of a test agent with appropriate negativecontrols (e.g., vehicle or diluent only and/or a composition or compoundknown in the art not to kill senescent cells) and appropriate positivecontrols. In vitro cell-based assays for characterizing senolytic agentsalso include controls for determining the effect of the agent onnon-senescent cells (e.g., quiescent cells or proliferating cells). Asenolytic agent reduces (i.e., decreases) percent survival of aplurality of senescent cells (i.e., in some manner reduces the quantityof viable senescent cells in the animal or in the cell-based assay)compared with one or more negative controls. Conditions for a particularin vitro assay include temperature, buffers (including salts, cations,media), and other components, which maintain the integrity of the testagent and reagents used in the assay, and which are familiar to a personskilled in the art and/or which can be readily determined.

The source of senescent cells for use in assays may be a primary cellculture, or culture adapted cell line, including but not limited to,genetically engineered cell lines that may contain chromosomallyintegrated or episomal recombinant nucleic acid sequences, immortalizedor immortalizable cell lines, somatic cell hybrid cell lines,differentiated or differentiatable cell lines, transformed cell lines,and the like. In a particular embodiment, the senescent cell is isolatedfrom biological sample obtained from a host or subject who has asenescent cell associated disease or disorder. In other embodiments,non-senescent cells, which may be obtained from a subject or may be aculture adapted line may be used and senescence induced by methodsdescribed herein and in the art, such as by exposure to irradiation or achemotherapeutic agent (e.g., doxorubicin). The biological sample may bea blood sample, biopsy specimen, body fluids (e.g., lung lavage,ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes,tissue explant, organ culture, or any other tissue or cell preparationfrom a subject. The sample may be a tissue or cell preparation in whichthe morphological integrity or physical state has been disrupted, forexample, by dissection, dissociation, solubilization, fractionation,homogenization, biochemical or chemical extraction, pulverization,lyophilization, sonication, or any other means for processing a samplederived from a subject or biological source. The subject may be a humanor non-human animal.

Transgenic animal models as described herein and in the art may be usedto determine killing or removal of senescent cells (see, e.g., Baker etal., supra; Nature, 479:232-36 (2011); Int'l Patent ApplicationPublication No. WO/2012/177927; Int'l Patent Application Publication No.WO 2013/090645). Exemplary transgenic animal models contain a transgenethat includes a nucleic acid that allows for controlled clearance ofsenescence cells (e.g., p16^(ink4a) positive senescent cells) as apositive control. The presence and level of senescent cells in thetransgenic animals can be determined by measuring the level of adetectable label or labels that are expressed in senescent cells of theanimal. The transgene nucleotide sequence includes a detectable label,for example, one or more of a red fluorescent protein; a greenfluorescent protein; and one or more luciferases to detect clearance ofsenescent cells.

Animal models that are described herein or in the art includesart-accepted models for determining the effectiveness of a senolyticagent to treat or prevent (i.e., reduce the likelihood of occurrence of)a particular senescence associated disease or disorder, such asatherosclerosis models, osteoarthritis models, COPD models, and IPFmodels. As described herein, pulmonary disease murine models, such as ableomycin pulmonary fibrosis model, and a chronic cigarette smokingmodel are applicable for diseases such as COPD and may be routinelypracticed by a person skilled in the art. Animal models for determiningthe effectiveness of a senolytic agent to treat and/or prevent (i.e.,reduce the likelihood of occurrence of) chemotherapy and radiotherapyside effect models or to treat or prevent (i.e., reduce the likelihoodof occurrence of) metastasis are described in International PatentApplication Publication Nos. WO 2013/090645 and WO 2014/205244, whichare incorporated herein by reference in their entirety. Animal modelsfor determining the effectiveness of agents for treating eye diseases,particularly age-related macular degeneration are also routinely used inthe art (see, e.g., Pennesi et al., Mol. Aspects Med. 33:487-509 (2012);Zeiss et al., Vet. Pathol. 47:396-413 (2010); Chavala et al., J. Clin.Invest. 123:4170-81 (2013)).

By way of non-limiting example and as described herein, osteoarthritisanimal models have been developed. Osteoarthritis may be induced in theanimal, for example, by inducing damage to a joint, for example, in theknee by surgical severing, incomplete or total, of the anterior cruciateligament. Osteoarthritis animal models may be used for assessing theeffectiveness of a senolytic agent to treat or prevent (i.e., reducingthe likelihood of occurrence of) osteoarthritis and cause a decrease inproteoglycan erosion and to induce (i.e., stimulate, enhance) collagen(such as collagen type 2) production, and to reduce pain in an animalthat has ACL surgery. Immunohistology may be performed to examine theintegrity and composition of tissues and cells in a joint.Immunochemistry and/or molecular biology techniques may also beperformed, such as assays for determining the level of inflammatorymolecules (e.g., IL-6) and assays for determining the level ofsenescence markers as noted above, using methods and techniquesdescribed herein and that may be routinely practiced by a person skilledin the art.

By way of another non-limiting example and as described herein,atherosclerosis animal models have been developed. Atherosclerosis maybe induced in the animal, for example, by feeding animals a high fatdiet or by using transgenic animals highly susceptible to developingatherosclerosis. Animal models may be used for determining theeffectiveness of a senolytic agent to reduce the amount of plaque or toinhibit formation of plaque in an atherosclerotic artery, to reduce thelipid content of an atherosclerotic plaque (i.e., reduce, decrease theamount of lipid in a plaque), and to cause an increase or to enhancefibrous cap thickness of a plaque. Sudan staining may be used to detectthe level of lipid in an atherosclerotic vessel. Immunohistology andimmunochemistry and molecular biology assays (e.g., for determining thelevel of inflammatory molecules (e.g., IL-6), and for determining thelevel of senescence markers as noted above), may all be performedaccording to methods described herein and that are routinely practicedin the art.

In still another example, and as described herein, mouse models in whichanimals are treated with bleomycin has been described (see, e.g., Penget al., PLoS One 2013; 8(4):e59348. doi: 10.1371/journal.pone.0059348.Epub 2013 Apr. 2; Mouratis et al., Curr. Opin. Pulm. Med. 17:355-61(2011)) for determining the effectiveness of an agent for treating IPF.In pulmonary disease animals models (e.g., a bleomycin animal model,smoke-exposure animal model, or the like), respiratory measurements maybe taken to determine elastance, compliance, static compliance, andperipheral capillary oxygen saturation (SpO₂). Immunohistology andimmunochemistry and molecular biology assays (e.g., for determining thelevel of inflammatory molecules (e.g., IL-6), and for determining thelevel of senescence markers as noted above), may all be performedaccording to methods described herein and that are routinely practicedin the art.

Determining the effectiveness of a senolytic agent to selectively killsenescent cells as described herein in an animal model may be performedusing one or more statistical analyses with which a skilled person willbe familiar. By way of example, statistical analyses such as two-wayanalysis of variance (ANOVA) may be used for determining the statisticalsignificance of differences between animal groups treated with an agentand those that are not treated with the agent (i.e., negative controlgroup, which may include vehicle only and/or a non-senolytic agent).Statistical packages such as SPSS, MINITAB, SAS, Statistika, Graphpad,GLIM, Genstat, and BMDP are readily available and routinely used by aperson skilled in the animal model art.

A person skilled in the art will readily appreciate that characterizinga senolytic agent and determining the level of killing by the senolyticagent can be accomplished by comparing the activity of a test agent withappropriate negative controls (e.g., vehicle only and/or a composition,agent, or compound known in the art not to kill senescent cells) andappropriate positive controls. In vitro cell-based assays forcharacterizing the agent also include controls for determining theeffect of the agent on non-senescent cells (e.g., quiescent cells orproliferating cells). A senolytic agent that is useful reduces (i.e.,decreases) percent survival of senescent cells (i.e., in some mannerreduces the quantity of viable senescent cells in the animal or in thecell-based assay) compared with one or more negative controls.Accordingly, a senolytic agent selectively kills senescent cellscompared with killing of non-senescent cells (which may be referred toherein as selectively killing senescent cells over non-senescent cells).In certain embodiments (either in an in vitro assay or in vivo (in ahuman or non-human animal)), the at least one senolytic agent kills atleast 20% of the senescent cells and kills no more than 5% ofnon-senescent cells. In other particular embodiments (either in an invitro assay or in vivo (in a human or non-human animal)), the at leastone senolytic agent kills at least about 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, or 65% of the senescent cells and kills no more than about 5%or 10% of non-senescent cells. In other particular embodiments (eitherin an in vitro assay or in vivo (in a human or non-human animal)), theat least one senolytic agent kills at least about 30%, 35%, 40%, 45%,50%, 55%, 60%, or 65% of the senescent cells and kills no more thanabout 5%, 10%, or 15% of non-senescent cells. In other particularembodiments (either in an in vitro assay or in vivo (in a human ornon-human animal)), the at least one senolytic agent kills at leastabout 40%, 45%, 50%, 55%, 60%, or 65% of the senescent cells and killsno more than about 5%, 10%, 15%, 20%, or 25% of non-senescent cells. Inother particular embodiments (either in an in vitro assay or in vivo (ina human or non-human animal)), the at least one senolytic agent kills atleast about 50%, 55%, 60%, or 65% of the senescent cells and kills nomore than about 5%, 10%, 15%, 20%, 25%, or 30% of non-senescent cells.Stated another way, a senolytic agent has at least 5-25, 10-50 or 10-100times (5×-25×, 10×-50× or 10×-100×) greater selectively for killingsenescent cells than for non-senescent cells (e.g., at least 5×, 10×,20×, 25×, 30×, 40×. 50×, 60×, 75×, 80×, 90×, or 100×). With respect tospecific embodiments of the methods described herein for treating asenescence-associated disease or disorder, the percent senescent cellskilled may refer to the percent senescent cells killed in a tissue ororgan that comprises senescent cells that contribute to onset,progression, and/or exacerbation of the disease or disorder. By way ofnon-limiting example, tissues of the brain, tissues and parts of theeye, pulmonary tissue, cardiac tissue, arteries, joints, skin, andmuscles may comprise senescent cells that may be reduced in percent asdescribed above by the senolytic agents described herein and therebyprovide a therapeutic effect. Moreover, selectively removing at least20% or at least 25% of senescent cells from an affected tissue or organcan have a clinically significant therapeutic effect. With respect tospecific embodiments of the methods described herein, such as fortreating a cardiovascular disease or disorder associated witharteriosclerosis, such as atherosclerosis, by administering a senolyticagent (i.e., in reference to vivo methods above), the percent senescentcells killed may refer to the percent senescent cells killed in anaffected artery containing plaque versus non-senescent cells killed inthe arterial plaque. In certain particular embodiments, in the methodsfor treating the cardiovascular disease, such as atherosclerosis, asdescribed herein, the at least one senolytic agent kills at least 20% ofthe senescent cells and kills no more than 5% of non-senescent cells inthe artery. In other particular embodiments, the senolytic agentselectively kills at least 25% of the senescent cells in thearteriosclerotic artery. In another embodiment, with respect to themethods described herein for treating osteoarthritis by administering asenolytic agent, the percent senescent cells killed may refer to thepercent senescent cells killed in an osteoarthritic joint versusnon-senescent cells killed in the osteoarthritic joint. In certainparticular embodiments, in the methods for treating osteoarthritis asdescribed herein, the at least one senolytic agent kills at least 20% ofthe senescent cells and kills no more than 5% of non-senescent cells inthe osteoarthritic joint. In other particular embodiments, the senolyticagent selectively kills at least 25% of the senescent cells in theosteoarthritic joint. In still another embodiment, with respect to themethods described herein for treating senescence associated pulmonarydisease or disorder (e.g., COPD, IPF) by administering at least onesenolytic agent, the percent senescent cells killed may refer to thepercent senescent cells killed in affected pulmonary tissue versusnon-senescent cells killed in the affected pulmonary tissue of the lung.In certain particular embodiments, in the methods for treatingsenescence associated pulmonary diseases and disorders as describedherein, a senolytic agent kills at least 20% of the senescent cells andkills no more than 5% of non-senescent cells in the affected pulmonarytissue. In other particular embodiments, the senolytic agent selectivelykills at least 25% of the senescent cells in the affected pulmonarytissue.

In certain embodiments, methods are provided for identifying (i.e.,screening for) agents that are useful senolytic agents for treating orpreventing (i.e., reducing the likelihood of occurrence of) a senescenceassociated disease or disorder. In one embodiment, a method foridentifying a senolytic agent for treating such diseases and disorders,comprises inducing cells to senesce to provide established senescentcells. Methods for inducing cells to senesce are described herein and inthe art and include, for example, exposure to radiation (e.g., 10 Gy istypically sufficient) or a chemotherapeutic agent (e.g., doxorubicin orother anthracycline). After exposure to the agent, the cells arecultured for an appropriate time and under appropriate conditions (e.g.,media, temperature, CO₂/O₂ level appropriate for a given cell type orcell line) to allow senescence to be established. As discussed herein,senescence of cells may be determined by determining any number ofcharacteristics, such as changes in morphology (as viewed by microscopy,for example); production of, for example, senescence-associatedβ-galactosidase (SA-β-gal), p161NK4a, p21, or any one or more SASPfactors (e.g., IL-6, MMP3). A sample of the senescent cells is thencontacted with a candidate agent (i.e., mixed with, combined, or in somemanner permitting the cells and the agent to interact). Persons skilledin the art will appreciate that the assay will include the appropriatecontrols, negative and positive, either historical or performedconcurrently. For example, a sample of control non-senescent cells thathave been cultured similarly as the senescent cells but not exposed to asenescence inducing agent are contacted with the candidate agent. Thelevel of survival of the senescent cells is determined and compared withthe level of survival of the non-senescent cells. A senolytic agent isidentified when the level of survival of the senescent cells is lessthan the level of survival of the non-senescent cells.

In a particular embodiment, the above described method to identify asenolytic agent may further comprise steps for identifying whether thesenolytic agent is useful for treating osteoarthritis. The method mayfurther comprise contacting the identified senolytic agent with cellscapable of producing collagen; and determining the level of collagenproduced by the cells. In particular embodiments, the cells arechondrocytes and the collagen is Type 2 collagen. The method may furthercomprise administering a candidate senolytic agent to a non-human animalwith arthritic lesions in a joint and determining one or more of (a) thelevel of senescent cells in the joint; (b) physical function of theanimal; (c) the level of one or more markers of inflammation; (d)histology of the joint; and (e) the level of Type 2 collagen produced,thereby determining therapeutic efficacy of the senolytic agent whereinone or more of the following is observed in the treated animal comparedwith an animal not treated with the senolytic agent: (i) a decrease inthe level of senescent cells in the joint of the treated animal; (ii)improved physical function of the treated animal; (iii) a decrease inthe level of one or more markers of inflammation in the treated animal;(iv) increased histological normalcy in the joint of the treated animal;and (v) an increase in the level of Type 2 collagen produced in thetreated animal. As described herein and in the art, the physicalfunction of the animal may be determined by techniques that determinethe sensitivity of a leg to an induced or natural osteoarthriticcondition, for example, by the animal's tolerance to bear weight on anaffected limb or the ability of the animal to move away from anunpleasant stimulus, such as heat or cold. Determining the effectivenessof an agent to kill senescent cells as described herein in an animalmodel may be performed using one or more statistical analyses with whicha skilled person will be familiar. Statistical analyses as describedherein and routinely practiced in the art may be applied to analyzedata.

In another particular embodiment, the above described method to identifya senolytic agent may further comprise steps for identifying whether thesenolytic agent is useful for treating a cardiovascular disease causedby or associated with arteriosclerosis. Accordingly, the method mayfurther comprise administering the senolytic candidate agent innon-human animals of in animal models for determining the effectivenessof an agent to reduce the amount of plaque, to inhibit formation ofplaque in an atherosclerotic artery, to reduce the lipid content of anatherosclerotic plaque (i.e., reduce, decrease the amount of lipid in aplaque), and/or to cause an increase or to enhance fibrous cap thicknessof a plaque. Sudan staining may be used to detect the level of lipid inan atherosclerotic vessel. Immunohistology, assays for determining thelevel of inflammatory molecules (e.g., IL-6), and/or assays fordetermining the level of senescence markers as noted above, may all beperformed according to methods described herein and routinely practicedin the art. In a specific embodiment, methods described herein foridentifying a senolytic agent may further comprise administering acandidate senolytic agent to a non-human animal with atheroscleroticplaque and determining one or more of (a) the level of senescent cellsin the artery; (b) physical function of the animal; (c) the level of oneor more markers of inflammation; (d) histology of the affected bloodvessel(s) (e.g., artery); and thereby determining therapeutic efficacyof the senolytic agent wherein one or more of the following is observedin the treated animal compared with an animal not treated with thesenolytic agent: (i) a decrease in the level of senescent cells in theartery of the treated animal; (ii) improved physical function of thetreated animal; (iii) a decrease in the level of one or more markers ofinflammation in the treated animal; (iv) increased histological normalcyin the artery of the treated animal. As described herein and in the art,the physical function of the animal may be determined by measuringphysical activity. Statistical analyses as described herein androutinely practiced in the art may be applied to analyze data.

In one embodiment, methods described herein for identifying a senolyticagent may comprise administering a candidate senolytic agent to anon-human animal pulmonary disease model such as a bleomycin model or asmoke-exposure animal model and determining one or more of (a) the levelof senescent cells in a lung; (b) lung function of the animal; (c) thelevel of one or more markers of inflammation; (d) histology of pulmonarytissue, thereby determining therapeutic efficacy of the senolytic agentwherein one or more of the following is observed in the treated animalcompared with an animal not treated with the senolytic agent: (i) adecrease in the level of senescent cells in the lungs and pulmonarytissue of the treated animal; (ii) improved lung function of the treatedanimal; (iii) a decrease in the level of one or more markers ofinflammation in the treated animal; and (iv) increased histologicalnormalcy in the pulmonary tissue of the treated animal. Respiratorymeasurements may be taken to determine elastance, compliance, staticcompliance, and peripheral capillary oxygen saturation (SpO₂). Lungfunction may be evaluated by determining any one of numerousmeasurements, such as expiratory reserve volume (ERV), forced vitalcapacity (FVC), forced expiratory volume (FEV) (e.g., FEV in one second,FEV1), FEV1/FEV ratio, forced expiratory flow 25% to 75%, and maximumvoluntary ventilation (MVVpeak expiratory flow (PEF), slow vitalcapacity (SVC). Total lung volumes include total lung capacity (TLC),vital capacity (VC), residual volume (RV), and functional residualcapacity (FRC). Gas exchange across alveolar capillary membrane can bemeasured using diffusion capacity for carbon monoxide (DLCO). Peripheralcapillary oxygen saturation (SpO₂) can also be measured. Statisticalanalyses as described herein and routinely practiced in the art may beapplied to analyze data.

The in vitro assays and in vivo assays (e.g., animal models) describedherein for identifying and characterizing senolytic agents may includeany one of the senolytic agents (e.g., Nutlin-3a, RG-7112, ABT-263,ABT-737, WEHI-539, A-1155463, MK-2206) described herein as positivecontrols. Conditions for a particular in vitro assay includetemperature, buffers (including salts, cations, media), and othercomponents (e.g., cells), which maintain the integrity of the test agentand reagents used in the assay, and which are familiar to a personskilled in the art and/or which can be readily determined. The assaysand techniques described herein may also be used for toxicologyanalytical methods, quality control assays, and the like that areroutinely performed during drug development and for quality assurance.Animal models for these methods and purposes may include non-humanprimate models, dog models, rodent models, or other animal modelsappropriate for determining the safety and efficacy of a senolyticagent.

Pharmaceutical Compositions

Also provided herein are pharmaceutical compositions that comprise asenolytic agent (e.g., a MDM2 inhibitor; an inhibitor of one or moreBcl-2 anti-apoptotic protein family members wherein the inhibitorinhibits at least Bcl-xL (e.g., a Bcl-xL selective inhibitor,Bcl-2/Bcl-xL/Bcl-w inhibitor, a Bcl-2/Bcl-xL or a Bcl-xL/Bcl-winhibitor); or an Akt specific inhibitor), as described herein and atleast one pharmaceutically acceptable excipient, which may also becalled a pharmaceutically suitable excipient or carrier (i.e., anon-toxic material that does not interfere with the activity of theactive ingredient). A pharmaceutical composition may be a sterileaqueous or non-aqueous solution, suspension or emulsion (e.g., amicroemulsion). The excipients described herein are examples and are inno way limiting. An effective amount or therapeutically effective amountrefers to an amount of the one or more senolytic agents administered toa subject, either as a single dose or as part of a series of doses,which is effective to produce a desired therapeutic effect.

When two or more senolytic agents are administered to a subject fortreatment of a disease or disorder described herein, each of thesenolytic agents may be formulated into separate pharmaceuticalcompositions. A pharmaceutical preparation may be prepared thatcomprises each of the separate pharmaceutical compositions (which may bereferred to for convenience, for example, as a first pharmaceuticalcomposition and a second pharmaceutical composition comprising each ofthe first and second senolytic agents, respectively). Each of thepharmaceutical compositions in the preparation may be administered atthe same time (i.e., concurrently) and via the same route ofadministration or may be administered at different times by the same ordifferent administration routes. Alternatively, two or more senolyticagents may be formulated together in a single pharmaceuticalcomposition.

In other embodiments, a combination of at least one senolytic agent andat least one inhibitor of an mTOR, NFκB, or PI3-k pathway may beadministered to a subject in need thereof. When at least one senolyticagent and an inhibitor of one or more of mTOR, NFκB, or PI3-k pathwaysare both used together in the methods described herein for selectivelykilling senescent cells, each of the agents may be formulated into thesame pharmaceutical composition or formulated in separate pharmaceuticalcompositions. A pharmaceutical preparation may be prepared thatcomprises each of the separate pharmaceutical compositions, which may bereferred to for convenience, for example, as a first pharmaceuticalcomposition and a second pharmaceutical composition comprising each ofthe senolytic agent and the inhibitor of one or more of mTOR, NFκB, orPI3-k pathways, respectively. Each of the pharmaceutical compositions inthe preparation may be administered at the same time and via the sameroute of administration or may be administered at different times by thesame or different administration routes.

In particular embodiments, a single senolytic agent is administered tothe subject and is the single (i.e., only, sole) active senolytic agent(i.e., monotherapy) used for treating the condition or disease. When asenolytic agent is the single senolytic agent, use of medications forother purposes such as palliative medications or medications that areused for comfort; or medications for treating a particular disease orcondition but that are not senolytic agents, such as drugs for loweringcholesterol or an eye wetting agent, and other such medications familiarto a person skilled in the medical art, are not necessarily excluded.Examples of other agents and medications that can be administered tosubjects with pulmonary diseases (e.g., COPD) include, by way ofnon-limiting example, bronchodilators (e.g., anti-cholinergics; beta-2agonists); pain relief medication; Agents and medications that can beadministered to subjects with osteoarthritis include hyaluronan, painrelievers (including topical medications), and steroids. Other agentsand medications that can be administered to subjects with acardiovascular disease include statins, beta blockers, nitroglyercin,aspirin.

Subjects may generally be monitored for therapeutic effectiveness usingassays and methods suitable for the condition being treated, whichassays will be familiar to those having ordinary skill in the art andare described herein. Pharmacokinetics of a senolytic agent (or one ormore metabolites thereof) that is administered to a subject may bemonitored by determining the level of the senolytic agent in abiological fluid, for example, in the blood, blood fraction (e.g.,serum), and/or in the urine, and/or other biological sample orbiological tissue from the subject. Any method practiced in the art anddescribed herein to detect the agent may be used to measure the level ofthe senolytic agent during a treatment course.

The dose of a senolytic agent described herein for treating a senescencecell associated disease or disorder may depend upon the subject'scondition, that is, stage of the disease, severity of symptoms caused bythe disease, general health status, as well as age, gender, and weight,and other factors apparent to a person skilled in the medical art.Pharmaceutical compositions may be administered in a manner appropriateto the disease to be treated as determined by persons skilled in themedical arts. In addition to the factors described herein and aboverelated to use of the senolytic agent for treating asenescence-associated disease or disorder, suitable duration andfrequency of administration of the senolytic agent may also bedetermined or adjusted by such factors as the condition of the patient,the type and severity of the patient's disease, the particular form ofthe active ingredient, and the method of administration. Optimal dosesof an agent may generally be determined using experimental models and/orclinical trials. The optimal dose may depend upon the body mass, weight,or blood volume of the subject. The use of the minimum dose that issufficient to provide effective therapy is usually preferred. Design andexecution of pre-clinical and clinical studies for a senolytic agent(including when administered for prophylactic benefit) described hereinare well within the skill of a person skilled in the relevant art. Whentwo or more senolytic agents are administered to treat asenescence-associated disease or disorder, the optimal dose of eachsenolytic agent may be different, such as less, than when either agentis administered alone as a single agent therapy. In certain particularembodiments, two senolytic agents in combination make actsynergistically or additively, and either agent may be used in a lesseramount than if administered alone. An amount of a senolytic agent thatmay be administered per day may be, for example, between about 0.01mg/kg and 100 mg/kg (e.g., between about 0.1 to 1 mg/kg, between about 1to 10 mg/kg, between about 10-50 mg/kg, between about 50-100 mg/kg bodyweight. In other embodiments, the amount of a senolytic agent that maybe administered per day is between about 0.01 mg/kg and 1000 mg/kg,between about 100-500 mg/kg, or between about 500-1000 mg/kg bodyweight. In particular embodiments, the total amount of an MDM2 inhibitor(e.g., Nutlin-3a), the total amount of the senolytic agent administeredper course of treatment each treatment cycle does not exceed 2100 mg/kg;in other embodiments, the total amount administered per course oftreatment does not exceed 1400 mg/kg. The optimal dose (per day or percourse of treatment) may be different for the senescence-associateddisease or disorder to be treated and may also vary with theadministrative route and therapeutic regimen.

Pharmaceutical compositions comprising a senolytic agent can beformulated in a manner appropriate for the delivery method by usingtechniques routinely practiced in the art. The composition may be in theform of a solid (e.g., tablet, capsule), semi-solid (e.g., gel), liquid,or gas (aerosol). In other certain specific embodiments, the senolyticagent (or pharmaceutical composition comprising same) is administered asa bolus infusion. In certain embodiments when the senolytic agent isdelivered by infusion, the senolytic agent is delivered to an organ ortissue comprising senescent cells to be killed via a blood vessel inaccordance with techniques routinely performed by a person skilled inthe medical art.

Pharmaceutical acceptable excipients are well known in thepharmaceutical art and described, for example, in Rowe et al., Handbookof Pharmaceutical Excipients: A Comprehensive Guide to Uses, Properties,and Safety, 5^(th) Ed., 2006, and in Remington: The Science and Practiceof Pharmacy (Gennaro, 21^(st) Ed. Mack Pub. Co., Easton, Pa. (2005)).Exemplary pharmaceutically acceptable excipients include sterile salineand phosphate buffered saline at physiological pH. Preservatives,stabilizers, dyes, buffers, and the like may be provided in thepharmaceutical composition. In addition, antioxidants and suspendingagents may also be used. In general, the type of excipient is selectedbased on the mode of administration, as well as the chemical compositionof the active ingredient(s). Alternatively, compositions describedherein may be formulated as a lyophilizate. A composition describedherein may be lyophilized or otherwise formulated as a lyophilizedproduct using one or more appropriate excipient solutions forsolubilizing and/or diluting the agent(s) of the composition uponadministration. In other embodiments, the agent may be encapsulatedwithin liposomes using technology known and practiced in the art. Incertain particular embodiments, a senolytic agent (e.g., ABT-263) is notformulated within liposomes for application to a stent that is used fortreating highly, though not totally, occluded arteries. Pharmaceuticalcompositions may be formulated for any appropriate manner ofadministration described herein and in the art.

A pharmaceutical composition may be delivered to a subject in needthereof by any one of several routes known to a person skilled in theart. By way of non-limiting example, the composition may be deliveredorally, intravenously, intraperitoneally, by infusion (e.g., a bolusinfusion), subcutaneously, enteral, rectal, intranasal, by inhalation,buccal, sublingual, intramuscular, transdermal, intradermal, topically,intraocular, vaginal, rectal, or by intracranial injection, or anycombination thereof. In certain particular embodiments, administrationof a dose, as described above, is via intravenous, intraperitoneal,directly into the target tissue or organ, or subcutaneous route. Incertain embodiments, a delivery method includes drug-coated or permeatedstents for which the drug is the senolytic agent. Formulations suitablefor such delivery methods are described in greater detail herein.

In certain particular embodiments, a senolytic agent (which may becombined with at least one pharmaceutically acceptable excipient to forma pharmaceutical composition) is administered directly to the targettissue or organ comprising senescent cells that contribute tomanifestation of the disease or disorder. In specific embodiments whentreating osteoarthritis, the at least one senolytic agent isadministered directly to an osteoarthritic joint (i.e.,intra-articularly) of a subject in need thereof. In other specificembodiments, a senolytic agent(s) may be administered to the joint viatopical, transdermal, intradermal, or subcutaneous route. In othercertain embodiments, methods are provided herein for treating acardiovascular disease or disorder associated with arteriosclerosis,such as atherosclerosis by administering directly into an artery. Inanother particular embodiment, a senolytic agent (which may be combinedwith at least one pharmaceutically acceptable excipient to form apharmaceutical composition) for treating a senescent-associatedpulmonary disease or disorder may be administered by inhalation,intranasally, by intubation, or intracheally, for example, to providethe senolytic agent more directly to the affected pulmonary tissue. Byway of another non-limiting example, the senolytic agent (orpharmaceutical composition comprising the senolytic agent) may bedelivered directly to the eye either by injection (e.g., intraocular orintravitreal) or by conjunctival application underneath an eyelid of acream, ointment, gel, or eye drops. In more particular embodiments, thesenolytic agent or pharmaceutical composition comprising the senolyticagent may be formulated as a timed release (also called sustainedrelease, controlled release) composition or may be administered as abolus infusion.

A pharmaceutical composition (e.g., for oral administration or forinjection, infusion, subcutaneous delivery, intramuscular delivery,intraperitoneal delivery or other method) may be in the form of aliquid. A liquid pharmaceutical composition may include, for example,one or more of the following: a sterile diluent such as water, salinesolution, preferably physiological saline, Ringer's solution, isotonicsodium chloride, fixed oils that may serve as the solvent or suspendingmedium, polyethylene glycols, glycerin, propylene glycol or othersolvents; antibacterial agents; antioxidants; chelating agents; buffersand agents for the adjustment of tonicity such as sodium chloride ordextrose. A parenteral composition can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic. Theuse of physiological saline is preferred, and an injectablepharmaceutical composition is preferably sterile. In another embodiment,for treatment of an ophthalmological condition or disease, a liquidpharmaceutical composition may be applied to the eye in the form of eyedrops. A liquid pharmaceutical composition may be delivered orally.

For oral formulations, at least one of the senolytic agents describedherein can be used alone or in combination with appropriate additives tomake tablets, powders, granules or capsules, and if desired, withdiluents, buffering agents, moistening agents, preservatives, coloringagents, and flavoring agents. The compounds may be formulated with abuffering agent to provide for protection of the compound from low pH ofthe gastric environment and/or an enteric coating. A senolytic agentincluded in a pharmaceutical composition may be formulated for oraldelivery with a flavoring agent, e.g., in a liquid, solid or semi-solidformulation and/or with an enteric coating.

A pharmaceutical composition comprising any one of the senolytic agentsdescribed herein may be formulated for sustained or slow release (alsocalled timed release or controlled release). Such compositions maygenerally be prepared using well known technology and administered by,for example, oral, rectal, intradermal, or subcutaneous implantation, orby implantation at the desired target site. Sustained-releaseformulations may contain the compound dispersed in a carrier matrixand/or contained within a reservoir surrounded by a rate controllingmembrane. Excipients for use within such formulations are biocompatible,and may also be biodegradable; preferably the formulation provides arelatively constant level of active component release. The amount ofactive agent contained within a sustained release formulation dependsupon the site of implantation, the rate and expected duration ofrelease, and the nature of the condition, disease or disorder to betreated or prevented.

In certain embodiments, the pharmaceutical compositions comprising asenolytic agent are formulated for transdermal, intradermal, or topicaladministration. The compositions can be administered using a syringe,bandage, transdermal patch, insert, or syringe-like applicator, as apowder/talc or other solid, liquid, spray, aerosol, ointment, foam,cream, gel, paste. This preferably is in the form of a controlledrelease formulation or sustained release formulation administeredtopically or injected directly into the skin adjacent to or within thearea to be treated (intradermally or subcutaneously). The activecompositions can also be delivered via iontophoresis. Preservatives canbe used to prevent the growth of fungi and other microorganisms.Suitable preservatives include, but are not limited to, benzoic acid,butylparaben, ethyl paraben, methyl paraben, propylparaben, sodiumbenzoate, sodium propionate, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, cetypyridinium chloride, chlorobutanol,phenol, phenylethyl alcohol, thimerosal, and combinations thereof.

Pharmaceutical compositions comprising a senolytic agent can beformulated as emulsions for topical application. An emulsion containsone liquid distributed the body of a second liquid. The emulsion may bean oil-in-water emulsion or a water-in-oil emulsion. Either or both ofthe oil phase and the aqueous phase may contain one or more surfactants,emulsifiers, emulsion stabilizers, buffers, and other excipients. Theoil phase may contain other oily pharmaceutically approved excipients.Suitable surfactants include, but are not limited to, anionicsurfactants, non-ionic surfactants, cationic surfactants, and amphotericsurfactants. Compositions for topical application may also include atleast one suitable suspending agent, antioxidant, chelating agent,emollient, or humectant.

Ointments and creams may, for example, be formulated with an aqueous oroily base with the addition of suitable thickening and/or gellingagents. Lotions may be formulated with an aqueous or oily base and willin general also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents, orcoloring agents. Liquid sprays may be delivered from pressurized packs,for example, via a specially shaped closure. Oil-in-water emulsions canalso be used in the compositions, patches, bandages and articles. Thesesystems are semisolid emulsions, micro-emulsions, or foam emulsionsystems.

In some embodiments, the senolytic agent(s) can be formulated witholeaginous bases or ointments to form a semisolid composition with adesired shape. In addition to the senolytic agent, these semisolidcompositions can contain dissolved and/or suspended bactericidal agents,preservatives and/or a buffer system. A petrolatum component that may beincluded may be any paraffin ranging in viscosity from mineral oil thatincorporates isobutylene, colloidal silica, or stearate salts toparaffin waxes. Absorption bases can be used with an oleaginous system.Additives may include cholesterol, lanolin (lanolin derivatives,beeswax, fatty alcohols, wool wax alcohols, low HLB(hydrophobellipophobe balance) emulsifiers, and assorted ionic andnonionic surfactants, singularly or in combination.

Controlled or sustained release transdermal or topical formulations canbe achieved by the addition of time-release additives, such as polymericstructures, matrices, that are available in the art. For example, thecompositions may be administered through use of hot-melt extrusionarticles, such as bioadhesive hot-melt extruded film. The formulationcan comprise a cross-linked polycarboxylic acid polymer formulation. Across-linking agent may be present in an amount that provides adequateadhesion to allow the system to remain attached to target epithelial orendothelial cell surfaces for a sufficient time to allow the desiredrelease of the compound.

An insert, transdermal patch, bandage or article can comprise a mixtureor coating of polymers that provide release of the active agents at aconstant rate over a prolonged period of time. In some embodiments, thearticle, transdermal patch or insert comprises water-soluble poreforming agents, such as polyethylene glycol (PEG) that can be mixed withwater insoluble polymers to increase the durability of the insert and toprolong the release of the active ingredients.

Transdermal devices (inserts, patches, bandages) may also comprise awater insoluble polymer. Rate controlling polymers may be useful foradministration to sites where pH change can be used to effect release.These rate controlling polymers can be applied using a continuouscoating film during the process of spraying and drying with the activecompound. In one embodiment, the coating formulation is used to coatpellets comprising the active ingredients that are compressed to form asolid, biodegradable insert.

A polymer formulation can also be utilized to provide controlled orsustained release. Bioadhesive polymers described in the art may beused. By way of example, a sustained-release gel and the compound may beincorporated in a polymeric matrix, such as a hydrophobic polymermatrix. Examples of a polymeric matrix include a microparticle. Themicroparticles can be microspheres, and the core may be of a differentmaterial than the polymeric shell. Alternatively, the polymer may becast as a thin slab or film, a powder produced by grinding or otherstandard techniques, or a gel such as a hydrogel. The polymer can alsobe in the form of a coating or part of a bandage, stent, catheter,vascular graft, or other device to facilitate delivery of the senolyticagent. The matrices can be formed by solvent evaporation, spray drying,solvent extraction and other methods known to those skilled in the art.

In certain embodiments of a method described herein for treating acardiovascular disease associated with or caused by arteriosclerosis,one or more senolytic agents may be delivered directly into a bloodvessel (e.g., an artery) via a stent. In a particular embodiment, astent is used for delivering a senolytic agent to an atheroscleroticblood vessel (an artery). A stent is typically a tubular metallicdevice, which has thin-metal screen-like scaffold, and which is insertedin a compressed form and then expanded at the target site. Stents areintended to provide long-term support for the expanded vessel. Severalmethods are described in the art for preparing drug-coated anddrug-embedded stents. For example, a senolytic agent may be incorporatedinto polymeric layers applied to a stent. A single type of polymer maybe used, and one or more layers of the senolytic agent permeated polymermay be applied to a bare metal stent to form the senolytic agent-coatedstent. The senolytic agent may also be incorporated into pores in themetal stent itself, which may also be referred to herein as a senolyticagent-permeated stent or senolytic agent-embedded stent. In certainparticular embodiments, a senolytic agent may be formulated withinliposomes and applied to a stent; in other particular embodiments, forexample, when the senolytic agent is ABT-263, the ABT-263 is notformulated in liposome. Placement of stents in an atherosclerotic arteryis performed by a person skilled in the medical art. A senolyticagent-coated or -embedded stent not only expands the affected bloodvessel (e.g., an artery) but also may be effective for one or more of(1) reducing the amount of plaque, (2) inhibiting formation of plaque,and (3) increasing stability of plaque (e.g., by decreasing lipidcontent of the plaque; and/or causing an increase in fibrous capthickness), particularly with respect to plaque proximal to the agentcoated or agent embedded stent.

In one particular embodiment, the senolytic agent administered to asubject who has an ophthalmic senescence associated or disease ordisorder may be delivered intraocularly or intravitreally. In otherspecific embodiments, a senolytic agent(s) may be administered to theeye by a conjunctival route, applying the senolytic agent to the mucousmembrane and tissues of the eye lid, either upper, lower, or both. Anyof these administrations may be bolus infusions. In other particularembodiments, a pharmaceutical composition comprising any one of thesenolytic agents described herein may be formulated for sustained orslow release (which may also be called timed release or controlledrelease), which formulations are described in greater detail herein. Incertain embodiments, methods are provided herein for treating orpreventing (i.e., reducing the likelihood of occurrence of; delaying theonset or development of, or inhibiting, retarding, slowing, or impedingprogression or severity of) an ocular disease, disorder, or condition(e.g., presbyopia, cataracts, macular degeneration); for selectivelykilling senescent cells in an eye of a subject, and/or inducing collagen(such as Type IV collagen) production in the eye of a subject in needthereof by administering at least one senolytic agent (which may becombined with at least one pharmaceutically acceptable excipient to forma pharmaceutical composition) directly to an eye.

For pharmaceutical compositions comprising a nucleic acid molecule, thenucleic acid molecule may be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid, and bacterial, viral and mammalian expression systems such as, forexample, recombinant expression constructs as provided herein.Techniques for incorporating DNA into such expression systems are wellknown to those of ordinary skill in the art. The DNA may also be“naked,” as described, for example, in Ulmer et al., Science259:1745-49, 1993 and reviewed by Cohen, Science 259:1691-92, 1993. Theuptake of naked DNA may be increased by coating the DNA ontobiodegradable beads, which are efficiently transported into the cells.Nucleic acid molecules may be delivered into a cell according to any oneof several methods described in the art (see, e.g., Akhtar et al.,Trends Cell Bio. 2:139 (1992); Delivery Strategies for AntisenseOligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al., Mol.Membr. Biol. 16:129-40 (1999); Hofland et al., Handb. Exp. Pharmacol.137:165-92 (1999); Lee et al., ACS Symp. Ser. 752:184-92 (2000); U.S.Pat. No. 6,395,713; Int'l Patent Appl. Publ. No. WO 94/02595); Selbo etal., Int. J. Cancer 87:853-59 (2000); Selbo et al., Tumour Biol.23:103-12 (2002); U.S. Patent Appl. Publ. Nos. 2001/0007666, and2003/077829).

Kits with unit doses of one or more of the agents described herein,usually in oral or injectable doses, are provided. Such kits may includea container containing the unit dose, an informational package insertdescribing the use and attendant benefits of the drugs in treating thesenescent cell associated disease, and optionally an appliance or devicefor delivery of the composition.

EXAMPLES Example 1 In Vitro Cell Assays for Determining SenolyticActivity of Nutlin-3a

Foreskin fibroblast cell lines HCA2 and BJ, lung fibroblast cell lineIMR90, and mouse embryonic fibroblasts were seeded in six-well platesand induced to senesce with 10 Gy of ionizing radiation (IR) or a 24 hrtreatment with doxorubicin (Doxo). Senescent phenotype was allowed todevelop for at least 7 days, at which point a cell count was made todetermine the baseline number of cells. Nutlin-3a treatment was theninitiated for a period of at least 9 days. Media alone or media withdrug as appropriate was refreshed at least every three days. At the endof the assay time period, cells are counted. Each condition was seededin three plate wells and counted independently. Initial cell countserves as a control to determine the induction of senescence, ascompared to the last day count without nutlin treatment. Initialnon-senescent cell count serves as a proxy to determine Nutlin-3atoxicity. FIG. 1 shows a schematic of the experiment design.

Foreskin fibroblast cell lines HCA2 and BJ, lung fibroblast cell lineIMR90, and mouse embryonic fibroblasts were exposed to 10 Gy of ionizingradiation (IR) to induce senescence. Seven days following irradiation,the cell were treated with varying concentrations of Nutlin-3a (0, 2.5μM, and 10 μM) for a period of 9 days, with the drug refreshed at leastevery 3 days. Percent survival was calculated as [cell count on day 9 ofNutlin-3a treatment/initial cell count on first day of Nutlin-3atreatment]. The results are shown in FIGS. 2A-D, which show thatNutlin-3a reduced cell survival of senescent foreskin fibroblasts (HCA2and BJ), lung fibroblasts (IMR90), and mouse embryonic fibroblasts(MEF), indicating Nutlin-3a is a senolytic agent.

Foreskin fibroblasts (HCA2) and aortic endothelial cells (Endo Aort)were treated with doxorubicin (250 nM) for one day (24 hours) to inducesenescence (see FIG. 1). Eight days following doxorubicin treatment,Nutlin-3a treatment was initiated. HCA2 cells were exposed to Nutlin-3afor 9 days, and aortic endothelial cells were exposed to Nutlin-3a for11 days. Media containing the compound or control media was refreshed atleast every 3 days. Percent survival was calculated as [cell count onthe last day of Nutlin-3a treatment/initial cell count on first day ofNutlin-3a treatment]. The results are shown in FIGS. 3A-B, which showthat doxorubicin-induced senescent cells are sensitive to Nutlin-3a.

Non-senescent foreskin fibroblasts (HCA2), lung fibroblasts (IMR90), andmouse embryonic fibroblasts (MEF) were treated with varyingconcentrations (0, 2.5 μM, and 10 μM) of Nutlin-3a for a period of 9days to assess Nutlin-3a toxicity. Cell counts were taken at the start(NS start) and end of Nutlin-3a treatment. The difference between countsof cells not treated with Nutlin-3a on day 9 (NS 0) and cell countsdetermined at day zero (NS start) reflects the cell growth over theindicated time period. The results are shown in FIGS. 4A-C, which showthat Nutlin-3a treatment reduces proliferation but is non-toxic tonon-senescent cells. Nutlin-3a treatment did not decrease the number ofcells below the starting level, indicating an absence of toxicity.Decrease in apparent survival between NS 0 and NS 2.5 and between NS 0and NS 10 reflects a decrease in cell growth. Without wishing to bebound by theory, Nutlin-3a may stabilize p53, leading to cell cyclegrowth arrest.

Non-senescent aortic endothelial (Endo Aort) cells and pre-adipocytes(Pread) were also treated with varying concentrations (0, 2.5 μM, and 10μM) of Nutlin-3a for a period of 11 days to assess Nutlin-3a toxicity,as described above. Cell counts were taken at the start at Day 0 (NSstart) and at the end of Nutlin-3a treatment (NS 0). The differencebetween counts of cells not treated with Nutlin-3a on day 11 (NS 0) andcell counts from NS start reflects the cell growth over the indicatedtime period. The results are shown in FIGS. 5A-B, illustrating thatNutlin-3a treatment reduces proliferation but is non-toxic tonon-senescent cells. As observed with fibroblasts, Nutlin-3a treatmentdoes not decrease the number of cells below the starting level,indicating an absence of toxicity. Decrease in apparent survival betweenNS 0 and NS 2.5 and between NS 0 and NS 10 reflects a decrease in cellgrowth.

Example 2 Nutlin-3A Treatment of p16-3MR Transgenic Mice

The capability of Nutlin-3a to remove senescent cells in vivo wasdetermined in transgenic p16-3MR mice (see, e.g., InternationalApplication Publication No. WO2013/090645). A schematic of theexperimental protocol is provided in FIG. 6. The transgenic mousecomprises a p16^(Ink4a) promoter operatively linked to a trimodal fusionprotein for detecting senescent cells and for selective clearance ofsenescent cells in these transgenic mice, which is illustrated in FIG.7. The promoter, p16^(Ink4a), which is transcriptionally active insenescent cells but not in non-senescent cells (see, e.g., Wang et al.,J. Biol. Chem. 276:48655-61 (2001); Baker et al., Nature 479:232-36(2011)), was engineered into a nucleic acid construct. 3MR (tri-modalityreporter) is a fusion protein containing functional domains of asynthetic Renilla luciferase (LUC), monomeric red fluorescence protein(mRFP), and truncated herpes simplex virus (HSV)-1 thymidine kinase(tTK), which allows killing by ganciclovir (GCV) (see, e.g., Ray et al.,Cancer Res. 64:1323-30 (2004)). The 3MR cDNA was inserted in frame withp16 in exon 2, creating a fusion protein containing the first 62 aminoacids of p16, but not a full-length wild-type p16 protein. Insertion ofthe 3MR cDNA also resulted in the occurrence of a stop codon in thep19^(ARF) reading frame in exon 2, thereby preventing full-lengthp19^(ARF) expression from the BAC as well. The p16^(Ink4a) gene promoter(approximately 100 kilobase pairs) was introduced upstream of anucleotide sequence encoding a trimodal reporter fusion protein.Alternatively, a truncated p16^(Ink4a) promoter may be used (see, e.g.,Baker et al., Nature, supra; International Application Publication No.WO2012/177927; Wang et al., supra). Thus, the expression of 3MR isdriven by the p16^(Ink4a) promoter in senescent cells only. Thedetectable markers, LUC and mRFP permitted detection of senescent cellsby bioluminescence and fluorescence, respectively. The expression of tTKpermitted selective killing of senescent cells by exposure to thepro-drug ganciclovir (GCV), which is converted to a cytotoxic moiety bytTK. Transgenic founder animals, which have a C57B16 background, wereestablished and bred using known procedures for introducing transgenesinto animals (see, e.g., Baker et al., Nature 479:232-36 (2011)).

Female C57/BL6 p16-3MR mice were randomized into doxorubicin+Nutlin-3atreated or doxorubicin only treated groups (see FIG. 6). Senescence wasinduced by intraperitoneal administration of doxorubicin at 10 mg/kg tothe mice ten days prior to administration of Nutlin-3a (Day −10).Nutlin-3a (25 mg/kg) was administered intraperitoneally daily from day10 to day 24 post-doxorubicin treatment (Group=9 mice). Control mice(doxorubicin treated) were injected with equal volumes of PBS (Group=3mice). Luminescence imaging (Xenogen Imaging system) was performed atDay 0 (i.e., 10 days post-doxorubicin treatment) as a baseline for eachmouse (100% intensity).

Luminescence imaging of the mice was performed on day 7, 14, 21, 28, and35 following the initiation of Nutlin-3a treatment. Reduction ofluminescence (L) was calculated as: L=(Imaging post-Nutlin-3atreatment)/(Baseline Imaging) %. If L is greater than or equal to 100%,the number of senescent cells was not reduced. If L is less than 100%,then the number of senescent cells was reduced. Every mouse wascalculated independently, and background was subtracted from eachsample. The results are presented in FIG. 8, which suggest thattreatment with Nutlin-3a reduced luminescence associated withdoxorubicin-induced senescence. A statistically significant decrease inluminescence was observed at day 14, day 28, and day 35 in Nutlin-3atreated animals.

Experiments were performed to determine the effect of Nutlin-3atreatment on expression of genes associated with senescence. Groups offemale C57/BL6 p16-3MR were treated as described above. Three weeksafter the end of Nutlin-3a treatment (day 35), the doxorubicin treatedmice (control) (N=3) and doxorubicin+Nutlin-3a-treated mice (N=6) weresacrificed. Skin and fat biopsies were collected for RNA extraction; fatbiopsies were collected for detection of senescence-associatedβ-galactosidase; and lungs were flash frozen in cryoprotectant OCT mediafor cryostat sectioning.

RNA was analyzed for mRNA levels of endogenous senescence markers (p21,p16^(INK4a) (p16), and p53) and SASP factors (mmp-3 and IL-6) relativeto actin mRNA (control for cDNA quantity) using the Roche UniversalProbe Library for real-time PCR assay. The results are presented inFIGS. 9A-E, which suggest Nutlin-3a treatment reduced expression of SASPfactors and senescence markers associated with doxorubicin-inducedsenescence. Values represent fold of induction of the respective mRNAover untreated control animals.

The frozen lung tissue were sectioned to 10 μM thickness and stainedwith primary rabbit polyclonal antibody against γH2AX (NovusBiologicals, LLC), which is a marker for double-strand breaks in cells(DNA damage). The sections were then stained with ALEXA FLUOR®dye-labeled secondary goat anti-rabbit antibody (Life Technologies) andcounterstained with 4′,6-diamidino-2-phenylindole (DAPI) (LifeTechnologies). The number of positive cells was calculated using ImageJimage processing program (National Institutes of Health, see Internet atimagej.nih.gov/ij/index.html) and represented as a percentage of thetotal number of cells. The results are presented in FIG. 10A-B, whichshow that nutlin-3A treatment reduced the number of cells with DNAdamage induced by doxorubicin. FIG. 10A shows reduced γH2AX staining indoxorubicin+Nutlin-3a treated cells compared with cells treated withdoxorubicin alone. FIG. 10B shows a reduction in the percent γH2AXpositive cells in doxorubicin+Nutlin-3a treated cells as compared tocells treated with doxorubicin alone.

Upon collection, fat biopsies were immediately fixed in 4% formalin andthen stained with a solution containing X-gal to detect the presence ofsenescence-associated-galactosidase (3-gal). Fat biopsies were incubatedovernight at 37° C. in X-gal solution and were photographed the nextday. Fat biopsies from untreated animals were used as a negative control(CTRL). The results are presented in FIG. 11, which show that Nutlin-3atreatment reduced senescence-associated 1-gal intensity in fat biopsiesfrom animals with doxorubicin-induced senescence similar to untreatednegative controls, as compared to mice treated with doxorubicin alone.

Example 3 MDM2 Inhibitor Removes Senescent Cells with Established SASP

Primary human fibroblast (IMR90) cells were induced to senesce byapplying 10Gy of irradiation. Seven days after irradiation (Day 0),cells were treated with 10 μM Nutlin-3a or vehicle (DMSO) for nine days(Day 9). The drug or vehicle was refreshed every three days.Drug/vehicle was removed at Day 9 and the cells were cultured for anadditional three days (Day 12). Cells were then fixed with 4%paraformaldehyde and stained by immunofluorescence with a specificanti-IL-6 antibody (R&D, AF-206-NA). Cells were counterstained with DAPIfor nuclear visualization. The percent IL-6 positive cells isillustrated in FIG. 12A. An example of IL-6 positive cellimmunofluorescence is shown in FIG. 12B. IL-6 positive cells weredetermined in an unbiased manner using CellProfiler software. Threedifferent cultures were assessed. Non-senescent cells had no detectablecells IL-6 production while senescent cells were about 8% positive atday 9 after vehicle (DMSO) treatment (16 days after irradiation).Nutlin-3a treatment decreased the percent IL-6 positive cells to a levelbelow 5%. At day 12, 3 days after Nutlin-3a was removed and 19 daysafter irradiation, IL-6 positive cells in the vehicle control were about9% and Nutlin3a treated cells were less than 1% positive for IL-6.

In another experiment, IMR90 cells were induced to senesce byirradiation (10 Gy). Seven days after irradiation, cells were treatedwith 10 μM Nutlin-3a or vehicle (DMSO) for nine days (Day 9). The drugor vehicle was refreshed every three days. Drug/vehicle was removed atDay 9 and the cells were cultured for an additional six days.Conditioned media from the treated cells was collected, and IL-6measurement by ELISA was performed (Perkin Elmer, AL223F). IL-6 levelsin culture media were determined by ELISA using a kit according tomanufacturer's instructions (AL223F, Perkin Elmer). Cells were fixedwith 4% paraformaldehyde and stained by immunofluorescence with aspecific anti-IL-6 antibody (R&D, AF-206-NA). The IL-6 level determinedby ELISA was normalized to the number of cells in each well. The dataare presented in FIG. 22C as a relative level of IL-6 in the treatedcells compared to the level in non-senescent cells (NS). The data arepresented as an average of three different cell samples.

The level of IL-6 in senescent cells was between 10-40 fold higher thanin non-senescent cells. Nutlin-3a treated senescent cells have a levelof IL-6 that is 5-9 fold lower than DMSO treated cells. Cells thatsurvive after Nutlin-3a treatment have a lower IL-6 secretion and byextrapolation, a lower SASP, suggesting that Nutlin-3a preferably killssenescent cells with a well-established SASP.

Example 4 MDM2 Inhibitor Removes Senescent Cells with Established SASP:SASP Factor Expression

Primary human fibroblast (IMR90) cells were induced to senesce byapplying 10Gy of irradiation. Seven days after irradiation (Day 0),cells were treated with 10 μM Nutlin-3a or vehicle (DMSO) for nine days(Day 9). The drug or vehicle was refreshed every three days.Drug/vehicle was removed at Day 9 and the cells were cultured for anadditional three days (Day 12) in media without drug or DMSO. Cells werethen collected, mRNA extracted, and cDNA prepared. Quantitative PCR(qPCR) was then performed to detect expression of various genes. Cellswere also collected at Day 12 after drug/vehicle had been removed forthree days. The data are presented as an average of three samples. Datawere normalized to actin and depicted as a ratio to non-senescent cells.The data are presented in FIGS. 13A-13F.

The level of p21 was approximately 10-fold greater in senescent cells,and was higher (approximately 90 fold) when cells were treated withNutlin-3a. Nutlin-3a stabilizes p53, and p53 is a transcription factoractivating the expression of the cyclin dependent kinase inhibitor p21.At day 12, the level of p21 in the DMSO treated cells was comparable tothe level at day 9, which was also comparable to the level in theNutlin-3a treated cells at day 12. These data suggest the acute effectof Nutlin-3a on cells is abrogated after three days after removal ofdrug exposure. The level of P16, another senescence marker, increased inirradiated cells and did not change in the presence of Nutlin-3a. Threedays after the drug has been removed (Day 12), a decrease in p16 levelwas observed. The level of IL-1a, a regulator of the SASP, decreasedonly after Nutlin-3a had been removed. CXCL-1, IL-6 and IL-8 are threeother SASP factors. The levels of all three were reduced when Nutlin-3awas present and remained lower after drug removal. These data show thatcells surviving Nutlin-3a treatment have a lower p16 level, suggestingthat Nutlin-3a preferably kills cells that are high p16 expressers.Similarly, SASP factors were reduced in surviving cells, also suggestingthat Nutlin-3a preferably kills cells with a higher SASP.

Example 5 MDM2 Inhibitor Removes Senescent Cells with Elevated DNADamage Response

Primary human fibroblast (IMR90) cells were induced to senesce byapplying 10Gy of irradiation. Seven days after irradiation (Day 0),cells were treated with 10 μM Nutlin-3a or vehicle (DMSO) for nine days(Day 9). The drug or vehicle was refreshed every three days.Drug/vehicle was removed at Day 9 and the cells were cultured for anadditional six days in media without drug or DMSO, changing media everythree days. Cells were collected at Day 0 (non-senescent cells), Day 9,Day 12, and Day 15, and protein extracted and processed forimmunoblotting (Western blotting). Two samples were processed at eachtime point; the results are provided for one sample in FIG. 14.

The data show that phosphorylation of the kinase ATM is lower in cellsthat have been treated with Nutlin-3a even when the drug has beenremoved (see pA™ S1981). Similarly, the substrate of ATM, H2AX, haddeclining levels of phosphorylation (see γH2AX) after Nutlin 3Atreatment and also after drug removal. In senescent cells, IkBa getsdegraded as the NF-kB pathway is activated, which leads to SASP. Thedata show that after drug is removed, the level of IkBa in Nutlin-3atreated cells approaches the level of IkBa in non-senescent cells. Thelevels of each of MDM2, p53 and p21 were elevated in the Nutlin-3atreated samples and decreased when the drug was removed.

These data also support that Nutlin-3a preferentially kills cells with ahigher SASP. In addition, because a lower level of activated ATM isproduced in surviving cells after drug treatment, these data suggestthat DNA damage response-activated senescent cells are the cells thatare sensitive to Nutlin-3a.

Example 6 Selective Toxicity of ABT-263 for Senescent Cells Using a CellCounting Assay

To determine whether ABT-263 is selectively toxic to senescent cellscompared to non-senescent cells, a cell counting assay was used todetermine cell survival following treatment with ABT-263. The generaltimelines and procedures for the cell counting assay are shown in FIG.15. IMR90 cells (human primary lung fibroblasts (IMR90) (IMR-90 (ATCC®CCL-186™, Mannassas, Va.) were seeded in six well plates, and cells wereinduced to senescence with 10 Gy of ionizing radiation (IR) (Day 0). Themedia was refreshed every 3 days. The senescent phenotype is allowed todevelop for 7 days at which point a cell count was made to determine thebaseline number of cells. In the senescent cells (irradiated) and thenon-senescent cells (the non-radiated cells), 3 μM ABT-263 wasintroduced into the media. Some cells were administered a media that didnot contain any ABT-263 as a control to account for any ABT-263toxicity. Each condition was seeded in three wells and countedindependently. Cells were counted after a 24 hour exposure to ABT-263(or control culture).

FIG. 16 demonstrates the effect of ABT-263 on non-senescent cells asmeasured as a percentage of survival of cells after 24 hours. Theaddition of ABT-263 to non-senescent (middle bar) did not decrease thecell growth below the starting level (left-most bar) indicating anabsence of toxicity in non-senescent cells. Non-ABT-263 treated cellsare shown as a control at the far-most right.

FIG. 17 demonstrates the effect of ABT-263 on senescent cells asmeasured as a percentage of survival of cells after 24 hours. Theaddition of ABT-263 to senescent cells (middle bar) had decreased cellgrowth below that of the starting level number of cells (left most bar).The ABT-263 treated cells had 28% of the cell counts before ABT-263treatment. Non-ABT-263 treated cells are shown as a control at thefar-most right.

Example 7 Selective Toxicity of ABT-263 for Senescent Cells Using ACellTiter-Glo® Cell Viability Assay

To determine whether ABT-263 is selectively toxic to senescent cellscompared to non-senescent cells, a cell viability assay was used toassess cell survival following treatment with ABT-263. The generaltimelines and procedures for the cell counting assay are shown in FIG.18. IMR90 cells (human primary lung fibroblasts (IMR90) (IMR-90 (ATCC®CCL-186™, Mannassas, Va.) were seeded in six well plates, and cells wereinduced to senescence with 10 Gy of ionizing radiation (IR) (Day 0). Themedia was refreshed every 3 days. The senescent phenotype is allowed todevelop for 7 days at which point a cell count was made to determine thebaseline number of cells followed by seeding into 96-well plates. On day8, the senescent cells (irradiated) and the non-senescent cells (thenon-radiated cells), were exposed to serial dilutions of ABT-263 for aperiod of 3 days. ABT-263 concentrations ranged from 0.5 nM to 3 μM.Each condition was seeded in triplicate.

After three days of treatment (Day 11), cells were assayed for cellsurvival using the commercially available CellTiter-Glo® (CTG)Luminescent Cell Viability Assay (Promega Corporation, Madison, Wis.).The assay determines the number of viable cells in culture based on thequantitation of ATP present which is an indicator of metabolicallyactive cells.

FIG. 19 shows IC50 curves of ABT-263 in senescent cells, and innon-senescent cells. The IC50 curve is a plot of the percentage of cellsurvival following treatment of ABT-263 as determined by the cellviability assay. The plot shows the effect of the various concentrationlevels of ABT-263 on cell survival. The IC50 of ABT-263 on non-senescentcells was 2.4 μM compared to an IC50 value of 140 nM on senescent cells,demonstrating the selective toxicity of ABT-263 for senescent cells. Anin vitro theoretical therapeutic index of 17 was observed.

Example 8 Assessment of Selective Toxicity of ABT-263 for SenescentCells of Various Cell Types

The methods of Example 7 were repeated in other cell strains. Cellstrains included Primary Renal Cortical Cells, ATCC Cat #PCS-400-011(FIG. 20), HCA2 foreskin fibroblast cells (FIG. 21), Primary SmallAirway Epithelial Cells, ATCC Cat #PCS-301-010 (lung) (FIG. 22), humanpooled Preadipocyte from patients (Pread) (FIG. 23), Mouse embryonicfibroblast extracted from C57BI6 mice (MEF) (FIG. 24), Primary CoronaryArtery Smooth Muscle, ATCC Cat #PCS-100-021 (Smth Mscl) (FIG. 25).

The experiments performed in these other cell strains were performedessentially as described in Example 7. As shown in FIG. 20, the IC50 ofABT-263 on non-senescent cells was 430 nM compared to an IC50 value of25 nM on senescent cells, demonstrating the selective toxicity ofABT-263 for senescent cells in renal epithelial cells.

As shown in FIG. 21, the IC50 of ABT-263 on non-senescent cells was nottoxic as up to 3 μM compared to an IC50 value of 410 nM on senescentcells, demonstrating the selective toxicity of ABT-263 for senescentcells in HCA2 cells.

Example 9

Assessment of Selective Toxicity of ABT-263 and Other Bcl-2 Inhibitorsfor Senescent Human Primary Lung Fibroblasts

To determine whether other Bcl-2 inhibitors demonstrate selectivetoxicity for senescent cells over non-senescent cells, cells weretreated with ABT-199 (Selleckem Cat #S8048, Houston, Tex.) or Obatoclax(Selleckem Cat #S1057). ABT-199 and Obatoclax are known Bcl-2inhibitors.

The experiments performed for assessing the effect of these other Bcl-2inhibitors were performed essentially as described in Example 7. Cellswere exposed to ABT-199 at serial dilution concentrations ranging from15 nM to 100 μM (FIGS. 26 and 27). Cells were exposed to Obatoclax atconcentrations ranging from 1.4 nM to 9 μM (FIG. 28).

As shown in FIGS. 26-27, ABT-199 had an IC50 value of 6 μM-15.8 μM innon-senescent cells compared to an IC50 value of 6.9 μM-12.4 μM insenescent cells. As shown in FIG. 28, Obatoclax had an IC50 value of 75nM in non-senescent cells compared to an IC50 value of 125 nM insenescent cells. FIG. 26-28 demonstrate the inability of ABT-199 andObatoclax to selectively target senescent cells over non-senescentcells.

A compound specific for Bcl-2A1 also did not selectively kill senescentcells. IMR90 cells were induced to senescence by irradiation asdescribed in Example 7. The irradiated IMR90 cells and non-senescentIMR90 cells were then exposed to a compound called ML214 that is aBcl-2A1 specific inhibitor. The level of killing of senescent cells wascomparable to the level of killing of non-senescent cells.

Example 10

Selective Toxicity for Senescent Cells of the Akt Inhibitor, Mk-2206Alone and in Combination with ABT-263

The effect of ABT-263 in combination with the Akt inhibitor MK-2206 wastested for selective toxicity of senescent cells compared tonon-senescent cells in IMR90 cells. The methods of Example 7 wererepeated except that cell cultures were exposed to 10 nM MK-2206(Selleckem, Cat #S1078) in addition to serial dilutions of ABT-263.

FIG. 29A shows the dose dependence plots of ABT-263 treatment incombination with 10 nM MK-2206 on senescent cells and non-senescentcells. ABT-263+MK-2206-treated senescent cells had an IC50 value of0.083 μM, whereas ABT-263+MK-2206 cells in non-senescent cells had anIC50 value>3 μM, yielding a selectivity index of >36 for senescentcells.

The senolytic effect of MK-2206 alone was determined by exposingsenescent IMR90 cells and non-senescent IMR90 cells (see procedures inExample 7) and to serial dilutions of MK-2206. The percent survival wasdetermined, and the results are present in FIG. 29B.

Example 11 An Animal Study for Determining the Senolytic Effect ofABT-263 in Mice

The senolytic effect of senolytic agents, e.g., ABT-263, can be assessedin animal models of senescence. An example of such an animal study isdescribed here. Senescence in animals can be induced through theadministration of doxorubicin followed by treatment of a senolyticagent. On day 35, mice are sacrificed, and fat and skin are collectedfor RNA analysis, while lungs are collected and flash frozen forimmunomicroscopy analysis. RNA is analyzed for expression of SASPfactors (mmp3, IL-6) and senescence markers (p21, p16, and p53). Frozenlung tissue is analyzed for DNA damage marker (γH2AX).

The mice to be tested contain a transgene insertion of p16-3MR. 3MR(tri-modality reporter) is a fusion protein containing functionaldomains of a synthetic Renilla luciferase (LUC), monomeric redfluorescence protein (mRFP), and truncated herpes simplex virus (HSV)-1thymidine kinase (tTK), which allows killing by ganciclovir (GCV). The3MR cDNA is inserted in frame with p16 in exon 2, creating a fusionprotein containing the first 62 amino acids of p16, but does not includethe full-length wild-type p16 protein. Insertion of the 3MR cDNA alsointroduces a stop codon in the p19^(ARF) reading frame in exon 2.

The effect of ABT-263 is analyzed by the reduction of luminescenceintensity. Female C57/B16 p16-3MR mice are treated with Doxorubicin.Luminescence is measured 10 days later and used as baseline for eachmouse (100% intensity). ABT-263 is administered intraperitoneally dailyfrom day 10 to day 24 post-doxorubicin treatment. Luminescence is thenmeasured at day 7, 14, 21, 28, 35 post-ABT-263 treatments, and finalvalues calculated as % of the baseline values. Control animals (DOXO)are injected with equal volume of PBS.

The level of mRNA of endogenous mmp-3, IL-6, p21, p16, and p53 in theskin and fat from animals after treatment with doxorubicin alone (DOXO)or doxorubicin plus ABT-263 is plotted. The values represent the foldinduction of the particular mRNA compared with untreated controlanimals.

Immunofluorescence microscopy of lung sections from doxorubicin treatedanimals (DOXO) and doxorubicin and ABT-263 can be detected by binding toa primary rabbit polyclonal antibody specific for γH2AX followed byincubation with a secondary goat anti-rabbit antibody, and thencounterstained with DAPI. The percent positive cells fromimmunofluorescence microscopy are calculated and can be represented aspercentage of the total number of cells. Data can be obtained fromdoxorubicin-treated mice (Doxo), and doxorubicin+ABT-263-treated mice).

ABT-263 can be analyzed for reduced senescence-associated (SA)β-galactosidase (β-gal) intensity of fat biopsies from animals firsttreated with doxorubicin. Female C57/BL6 p16-3MR mice are treated withdoxorubicin. A portion of the doxorubicin treated animals receiveABT-263or PBS (DOXO) daily from day 10 to day 24 post-doxorubicintreatment. Three weeks after the ABT-263 treatment, mice are sacrificedand fat biopsies immediately fixed and stained with a solutioncontaining X-Gal. Untreated animals are used as negative control (CTRL).

Example 12 In Vitro Cell Assays for Determining Senolytic Activity ofWEHI-539

Lung fibroblast cell line IMR90 (human primary lung fibroblasts, ATCC®CCL-186™, Manassas, Va.) and a renal cell line (Primary Renal CorticalCells, ATCC Cat. No. PCS-400-011) were seeded in six-well plates andinduced to senesce with 10 Gy of ionizing radiation (IR). Senescentphenotype was allowed to develop for at least 7 days.

After senescence phenotype had developed, cells were re-seeded into 96well plates, and senescent cells (irradiated) and non-senescent cells(the non-radiated cells), were exposed to three-fold serial dilutions ofWEHI-539 for a period of 3 days. WEHI-539 concentrations ranged from0.0075 μM to 15 μM. After the three days, cell survival was determinedusing the commercially available CellTiter-Glo® Luminescent CellViability Assay (Promega Corporation, Madison, Wis.). The assaydetermines the number of viable cells in culture based on thequantitation of ATP present which is an indicator of metabolicallyactive cells. FIG. 30 presents the IMR90 cell survival (see FIG. 30A)and renal cell survival (see FIG. 30B).

Example 13 WEHI-539 Treatment of p16-3MR Transgenic Mice

This example describes an animal model useful for determining thecapability of a senolytic agent to selectively kill senescent cells invivo. The capability of WEHI-539 or another senolytic agent to removesenescent cells in vivo is determined in transgenic p16-3MR mice (see,e.g., International Application Publication No. WO2013/090645). Anexperiment is performed in a similar manner to the procedureillustration in the schematic provided in FIG. 6. The transgenic mousecomprises a p16^(Ink4a) promoter operatively linked to a trimodal fusionprotein for detecting senescent cells and for selective clearance ofsenescent cells in these transgenic mice, which is illustrated in FIG.7. The promoter, p16^(Ink4a), which is transcriptionally active insenescent cells but not in non-senescent cells (see, e.g., Wang et al.,J. Biol. Chem. 276:48655-61 (2001); Baker et al., Nature 479:232-36(2011)), was engineered into a nucleic acid construct. 3MR (tri-modalityreporter) is a fusion protein containing functional domains of asynthetic Renilla luciferase (LUC), monomeric red fluorescence protein(mRFP), and truncated herpes simplex virus (HSV)-1 thymidine kinase(tTK), which allows killing by ganciclovir (GCV) (see, e.g., Ray et al.,Cancer Res. 64:1323-30 (2004)). The 3MR cDNA was inserted in frame withp16 in exon 2, creating a fusion protein containing the first 62 aminoacids of p16, but not a full-length wild-type p16 protein. Insertion ofthe 3MR cDNA also resulted in the occurrence of a stop codon in thep19^(ARF) reading frame in exon 2, thereby preventing full-lengthp19^(ARF) expression from the BAC as well. The p16^(Ink4a) gene promoter(approximately 100 kilobase pairs) was introduced upstream of anucleotide sequence encoding a trimodal reporter fusion protein.Alternatively, a truncated p16^(Ink4a) promoter may be used (see, e.g.,Baker et al., Nature, supra; International Application Publication No.WO2012/177927; Wang et al., supra). Thus, the expression of 3MR isdriven by the p16^(Ink4a) promoter in senescent cells only. Thedetectable markers, LUC and mRFP permitted detection of senescent cellsby bioluminescence and fluorescence, respectively. The expression of tTKpermitted selective killing of senescent cells by exposure to thepro-drug ganciclovir (GCV), which is converted to a cytotoxic moiety bytTK. Transgenic founder animals, which have a C57B16 background, wereestablished and bred using known procedures for introducing transgenesinto animals (see, e.g., Baker et al., Nature 479:232-36 (2011)).

To determine the senolytic activity of an agent, such as WEHI-539,female C57/BL6 p16-3MR mice are randomized into doxorubicin+WEHI-539treated or doxorubicin only treated groups. Senescence is induced byintraperitoneal administration of doxorubicin at 10 mg/kg to the miceten days prior to administration of WEHI-539 (Day −10). WEHI-539 isadministered intraperitoneally daily from day 10 to day 24post-doxorubicin treatment (Group=9 mice). Control mice (doxorubicintreated) are injected with equal volumes of PBS (Group=3 mice).Luminescence imaging (Xenogen Imaging system) is performed at Day 0(i.e., 10 days post-doxorubicin treatment) as a baseline for each mouse(100% intensity).

Luminescence imaging of the mice is performed on day 7, 14, 21, 28, and35 following the initiation of WEHI-539 treatment. Reduction ofluminescence (L) is calculated as: L=(Imaging post-WEHI-539treatment)/(Baseline Imaging) %. If L is greater than or equal to 100%,the number of senescent cells was not reduced. If L is less than 100%,then the number of senescent cells was reduced. Every mouse iscalculated independently, and background is subtracted from each sample.

Experiments are performed to determine the effect of WEHI-539 treatmenton expression of genes associated with senescence. Groups of femaleC57/BL6 p16-3MR are treated as described above. Three weeks after theend of WEHI-539 treatment (day 35), the doxorubicin treated mice(control) (N=3) and doxorubicin+WEHI-539-treated mice (N=6) aresacrificed. Skin and fat biopsies are collected for RNA extraction; fatbiopsies are collected for detection of senescence-associatedβ-galactosidase; and lungs are flash frozen in cryoprotectant OCT mediafor cryostat sectioning.

RNA is analyzed for mRNA levels of endogenous senescence markers (e.g.,p21, p16^(INK4a) (p16), and p53) and SASP factors (e.g., mmp-3 and IL-6)relative to actin mRNA (control for cDNA quantity) using the RocheUniversal Probe Library for real-time PCR assay.

The frozen lung tissue is sectioned to 10 μM thickness and stained withprimary rabbit polyclonal antibody against γH2AX (Novus Biologicals,LLC), which is a marker for double-strand breaks in cells (DNA damage).The sections are then stained with ALEXA FLUOR® dye-labeled secondarygoat anti-rabbit antibody (Life Technologies) and counterstained with4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies). The number ofpositive cells is calculated using ImageJ image processing program(National Institutes of Health, see Internet atimagej.nih.gov/ij/index.html) and represented as a percentage of thetotal number of cells.

Upon collection, fat biopsies are immediately fixed in 4% formalin andthen stained with a solution containing X-gal to detect the presence ofsenescence-associated β-galactosidase (β-gal). Fat biopsies areincubated overnight at 37° C. in X-gal solution and are photographed thenext day. Fat biopsies from untreated animals are used as a negativecontrol (CTRL).

Example 14 Capability of BCL-XL Inhibitor to Remove Senescent Cells withEstablished SASP

This example describes a method for determining the effect of asenolytic agent on killing of senescent cells that have establishedSASP. Primary human fibroblast (IMR90) cells are induced to senesce byapplying 10Gy of irradiation. Seven days after irradiation (Day 0),cells are treated with 10 μM of a BCL-XL inhibitor (e.g., WEHI-539) or aBCL-2/BCL-XL inhibitor or vehicle (DMSO) for nine days (Day 9). The drugor vehicle is refreshed every three days. Drug/vehicle is removed at Day9 and the cells are cultured for an additional three days (Day 12).Cells are then fixed with 4% paraformaldehyde and stained byimmunofluorescence with a specific anti-IL-6 antibody (R&D, AF-206-NA).Cells are counterstained with DAPI for nuclear visualization. IL-6positive cells are determined in an unbiased manner using CellProfilersoftware.

In another experiment, IMR90 cells are induced to senesce by irradiation(10 Gy). Seven days after irradiation, cells are treated with senolyticagent (e.g., a BCL-XL inhibitor (e.g., WEHI-539) or a BCL-2/BCL-XLinhibitor; MDM2 inhibitor; Akt inhibitor) or vehicle (DMSO) for ninedays (Day 9). The drug or vehicle is refreshed every three days.Drug/vehicle is removed at Day 9 and the cells are cultured for anadditional six days. Conditioned media from the treated cells iscollected, and IL-6 measurement by ELISA is performed (Perkin Elmer,AL223F). IL-6 levels in culture media are determined by ELISA using akit according to manufacturer's instructions (AL223F, Perkin Elmer).Cells are fixed with 4% paraformaldehyde and stained byimmunofluorescence with a specific anti-IL-6 antibody (R&D, AF-206-NA).The IL-6 level determined by ELISA is normalized to the number of cellsin each well.

Example 15 Capability of a Senolytic Agent to Remove Senescent Cellswith Established SASP: SASP Factor Expression

This example describes a method for determining the effect of asenolytic agent on SASP factor expression. Primary human fibroblast(IMR90) cells are induced to senesce by applying 10Gy of irradiation.Seven days after irradiation (Day 0), cells are treated with a senolyticagent (e.g., a BCL-XL inhibitor (e.g., WEHI-539) or a BCL-2/BCL-XLinhibitor; MDM2 inhibitor; Akt inhibitor) or vehicle (DMSO) for ninedays. The drug or vehicle is refreshed every three days. Afterdrug/vehicle is removed prior to evaluation of SASP expression at Day 9,the cells are cultured for an additional three days in media withoutdrug or DMSO. Cells are then collected, mRNA extracted, and cDNAprepared. Quantitative PCR (qPCR) is then performed to detect expressionof various genes. Cells are also collected at Day 12 after drug/vehiclehad been removed for three days. Data are normalized to actin anddepicted as a ratio to non-senescent cells.

Example 16 Capability of a Senolytic Agent to Remove Senescent Cellswith Elevated DNA Damage Response

This example describes a method for determining the effect of asenolytic agent on selectively killing senescent that that have anelevated DNA damage response. Primary human fibroblast (IMR90) cells areinduced to senesce by applying 10Gy of irradiation. Seven days afterirradiation (Day 0), cells are treated with a senolytic agent (forexample, a BCL-XL inhibitor (e.g., WEHI-539) or a BCL-2/BCL-XLinhibitor, MDM2 inhibitor; Akt inhibitor) or vehicle (DMSO) for ninedays (Day 9). The drug or vehicle is refreshed every three days.Drug/vehicle is removed at Day 9 and the cells are cultured for anadditional six days in media without drug or DMSO, changing media everythree days. Cells are collected at Day 0 (non-senescent cells), Day 9,Day 12, and Day 15, and protein extracted and processed forimmunoblotting (Western blotting). Two samples are processed at eachtime point.

Example 17 BCL-XL Selective Inhibitor Kills Senescent Cells ViaApoptosis

Lung fibroblast cell line IMR90 (human primary lung fibroblasts, ATCC®CCL-186™, Manassas, Va.) were seeded in six-well plates and induced tosenesce with 10 Gy of ionizing radiation (IR) as described in Example12. After senescence was established, cells were re-seeded into 96 wellplates. The pan-caspase inhibitor Q-VD-OPh (20 μM) was added to wells ofsenescent cells (irradiated) (IMR90 Sen(IR)) and to wells containingnon-senescent cells (the non-radiated cells) (IMR90 NS). Four hourslater, the senescent and non-senescent cells were each exposed for aperiod of 3 days to 1.67 or 5 μM WEHI-539. At the end of the assay timeperiod, cells were counted. Each condition was seeded in three platewells and counted independently. Initial cell count served as a controlto determine the induction of senescence, as compared to the last daycount without WEHI-539 treatment. Initial non-senescent cell countserves as a proxy to determine WEHI-539 toxicity. Cell survival wasdetermined using the commercially available CellTiter-Glo® LuminescentCell Viability Assay (Promega Corporation, Madison, Wis.). The assaydetermines the number of viable cells in culture based on thequantitation of ATP present which is an indicator of metabolicallyactive cells. FIG. 31 (left side) is an illustration that WEHI-539selectively kills senescent cells (see Example 12) and illustrates theWEHI-539 concentrations used in this experiment. In the presence of thepan-caspase inhibitor, the percent of surviving senescent cellsincreased (FIG. 31, right side).

Example 18 Effective Killing of Senescent Cells by Inhibiting BCL-XL

This example demonstrates that BCL-XL is the BCL-2 anti-apoptotic familymember important for apoptosis of senescent cells. Short hairpin RNAs(shRNA) comprising sequences specific for BCL-2, BCL-XL (also calledBCL2L1), and BCL-w (also called BCL2L2) were prepared and introducedinto lentiviral vectors. Four different shRNAs for each of BCL-XL andBCL-w and three for BCL-2 were synthesized by the Broad Institute of MITand Harvard (Cambridge, Mass.). Lentiviral vectors comprising eachrespective shRNA were purchased from Sigma Aldrich (St. Louis, Mo.). TheshRNA sequences and the target sequences are provided in the tablebelow. The nucleotide sequence of each protein can be readily obtainedfrom public databases (see, e.g., Bcl-xL at GenBank NM_001191.2 andNM_138578.1 (BCL2-like 1 (BCL2L1)); Bcl-xL at GenBank NM_004050.3(BCL2-like 2 (BCL2L2)); and Bcl-2 at NM_000633.2, NM_000657 (B-cellCLL/lymphoma 2 (BCL2)).

Triplicate samples of senescent cells and non-senescent cells weretransduced with each of the different lentiviral vectors and with twocontrol vectors according to methods practiced in the art. Controlsamples include senescent and non-senescent cells that were nottransduced (NT) with a lentivirus. IMR90 cells were induced to senesceby exposure to 10 Gy of ionizing radiation (IR) as described in Example12. After senescence phenotype had developed, cells were re-seeded into96 well plates, and shRNA was added. After 24 hrs, the shRNA was removedand media was refreshed. Media was again refreshed after 3 days. Afterthe last media refresh (6 days after shRNA removal), survival wasmeasured with CellTiter-Glo® Luminescent Cell Viability Assay.

TABLE shRNA Sequences Protein SYMBOL Encoded shRNA SequenceTarget Sequence BCL2 Bcl-2 CCGGCCGGGAGATAGTGATGAAGTACTC CCGGGAGATAGTGAGTACTTCATCACTATCTCCCGGTTTTT GATGAAGTA G (SEQ ID NO: 2) (SEQ ID NO: 1)BCL2 Bcl-2 CCGGGTGATGAAGTACATCCATTATCTC GTGATGAAGTACAGAGATAATGGATGTACTTCATCACTTTTT TCCATTAT G (SEQ ID NO: 4) (SEQ ID NO: 3)BCL2 Bcl-2 CCGGGTGATGAAGTACATCCATTATCTC GTGATGAAGTACAGAGATAATGGATGTACTTCATCACTTTTT TCCATTAT G (SEQ ID NO: 4) (SEQ ID NO: 3)BCL2 Bcl-2 CCGGAGAGTGACAGTGGATTGCATTCTC AGAGTGACAGTGGAGAATGCAATCCACTGTCACTCTTTTTT GATTGCATT G (SEQ ID NO: 6) (SEQ ID NO: 5)BCL2L1 Bcl-xL CCGGGCTCACTCTTCAGTCGGAAATCTC GCTCACTCTTCAGGAGATTTCCGACTGAAGAGTGAGCTTTTT TCGGAA G (SEQ ID NO: 8) (SEQ ID NO: 7)BCL2L1 Bcl-xL CCGGGTGGAACTCTATGGGAACAATCTC GTGGAACTCTATGGAGATTGTTCCCATAGAGTTCCACTTTTT GGAACA G (SEQ ID NO: 10) (SEQ ID NO: 9)BCL2L1 Bcl-xL CCGGGTTTAGTGATGTGGAAGAGAACTC GTTTAGTGATGTGGAGTTCTCTTCCACATCACTAAACTTTTT GAAGAG G (SEQ ID NO: 12) (SEQ ID NO: 11)BCL2L1 Bcl-xL CCGGGCTCACTCTTCAGTCGGAAATCTC GCTCACTCTTCAGGAGATTTCCGACTGAAGAGTGAGCTTTTT TCGGAAAT G (SEQ ID NO: 14) (SEQ ID NO: 13)BCL2L2 Bcl-w CCGGTGGCAGACTTTGTAGGTTATACTC TGGCAGACTTTGTGAGTATAACCTACAAAGTCTGCCATTTTT AGGTTA G (SEQ ID NO: 16) (SEQ ID NO: 15)BCL2L2 Bcl-w CCGGGTCAACAAGGAGATGGAACCACTC GTCAACAAGGAGGAGTGGTTCCATCTCCTTGTTGACTTTTT ATGGAAC G (SEQ ID NO: 18) (SEQ ID NO: 17)BCL2L2 Bcl-w CCGGCAGAAGGGTTATGTCTGTGGACTC CAGAAGGGTTATGAGTCCACAGACATAACCCTTCTGTTTTT GTCTGTG G (SEQ ID NO: 20) (SEQ ID NO: 19)BCL2L2 Bcl-w CCGGCCATTAGATGAGTGGGATTTACTC CCATTAGATGAGTGAGTAAATCCCACTCATCTAATGGTTTTT GGGATTTA TG (SEQ ID NO: 22)(SEQ ID NO: 21)

Survival of senescent cells and non-senescent cells was then determinedin triplicate for each shRNA tested. The shRNAs as listed in order inthe table are represented in the figure from left to right. The secondand third shRNA sequences specific for BCL-2 are identical. The ratio ofsenescent cell survival to non-senescent cell survival is presented foreach shRNA in FIG. 32. A ratio of 1.0 indicates no difference in theproportion of survival of senescent cells compared with non-senescentcells. Introduction of three of the four BCL-XL specific shRNA moleculesinto senescent cells resulted in significant senescent cell deathcompared with senescent cells into which Bcl-w or BCL-2 specific shRNAswere introduced. The data illustrate that BCL-XL expression is importantto survival of senescent cells.

Example 19 Effective Killing of Senescent Cells by Inhibiting Bcl-2Anti-Apoptotic Protein Family Members

To determine whether other Bcl-2/Bcl-xL/Bcl-xL inhibitors areselectively toxic to senescent cells compared to non-senescent cells, acell viability assay was used to assess cell survival followingtreatment with ABT-737. The general timelines and procedures for thecell counting assay are shown in FIG. 18 and described in Example 7.IMR90 cells (human primary lung fibroblasts) were seeded in six wellplates, and cells were induced to senescence with 10 Gy of ionizingradiation (IR) (Day 0). The media was refreshed every 3 days. Thesenescent phenotype is allowed to develop for 7 days at which point acell count was made to determine the baseline number of cells followedby seeding into 96-well plates. On day 8, the senescent cells(irradiated) and the non-senescent cells (the non-radiated cells), wereexposed to serial dilutions of ABT-737 for a period of 3 days. ABT-737concentrations were serially diluted starting at 50 μM. Each conditionwas seeded in triplicate.

After three days of treatment (Day 11), cells were assayed for cellsurvival using CellTiter-Glo® (CTG) Luminescent Cell Viability Assay.The assay determines the number of viable cells in culture based on thequantitation of ATP present, which is an indicator of metabolicallyactive cells.

FIG. 33 shows IC50 curves of ABT-737 in senescent cells and innon-senescent cells. The IC50 curve is a plot of the percentage of cellsurvival following treatment of ABT-737 as determined by the cellviability assay. The plot shows the effect of the various concentrationlevels of ABT-737 on cell survival.

Example 20 BCL-2/BCL-xL/BCL-w Inhibitor Kills Senescent Cells ViaApoptosis

An experiment as described in Example 17 was performed to determinewhether other inhibitors of one or more BCL-2 anti-apoptotic familymembers kill senescent cells by apoptosis. Lung fibroblast cell lineIMR90 (human primary lung fibroblasts, ATCC® CCL-186™, Manassas, Va.)were seeded in six-well plates and induced to senesce with 10 Gy ofionizing radiation (IR) as described in Example 12. After senescence wasestablished, cells were re-seeded into 96 well plates. The pan-caspaseinhibitor Q-VD-OPh (20 μM) was added to wells of senescent cells(irradiated) (IMR90 Sen(IR)) and to wells containing non-senescent cells(the non-radiated cells) (IMR90 NS). Four hours later, the senescent andnon-senescent cells were each exposed for a period of 3 days to 0.33 or1 μM ABT-263 (Navitoclax). At the end of the assay time period, cellswere counted. Each condition was seeded in three plate wells and countedindependently. Initial cell count served as a control to determine theinduction of senescence, as compared to the last day count withoutABT-263 treatment. Initial non-senescent cell count serves as a proxy todetermine ABT-263 toxicity. Cell survival was determined usingCellTiter-Glo® Luminescent Cell Viability Assay (Promega Corporation,Madison, Wis.). The assay determines the number of viable cells inculture based on the quantitation of ATP present which is an indicatorof metabolically active cells. FIG. 34 (top graphic) is an illustrationthat ABT-263 selectively kills senescent cells and illustrates theABT-263 concentrations used in this experiment. In the presence of thepan-caspase inhibitor, the percent of surviving senescent cellsincreased (FIG. 34, lower graphic).

Example 21 Effect of Removal of Senescent Cells in Animal Model ofOsteoarthritis

A table and schematic of two osteoarthritis mouse model study designsare presented in FIGS. 35 and 36, respectively. The two treatmentstudies were designed to determine the effect of removing senescentcells in an animal model of osteoarthritis.

Parallel studies were performed. One study investigated the effect ofeliminating senescent cells with ganciclovir (GCV) in 3MR mice. Miceunderwent surgery to cut the anterior cruciate ligament of one rear limbto induce osteoarthritis in the joint of that limb. During week 2post-surgery, 3MR mice received 2.5 μg GCV to the operated knee byintra-articular injection, qd for 5 days, with a 2^(nd) treatment (2.5μg GCV qd for 5 days) during week 4 post-surgery. At the end of 4 weekspost-surgery, operated joints of the mice were monitored for presence ofsenescent cells, assessed for function, monitored for markers ofinflammation, and underwent histological assessment.

In a parallel study, C57BL/6J mice underwent surgery to cut the anteriorcruciate ligament of one rear limb to induce osteoarthritis in the jointof that limb. During week 3 and week 4 post-surgery, the mice weretreated with 5.8 μg of Nutlin-3A (n=7) per operated knee byintra-articular injection, qod for 2 weeks. At the end of 4 weekspost-surgery, joints of the mice were monitored for presence ofsenescent cells, assessed for function, monitored for markers ofinflammation, and underwent histological assessment.

Two control groups of mice were included in the studies performed: onegroup comprising C57BL/6J or 3MR mice that had undergone a sham surgery(n=3) (i.e., surgical procedures followed except for cutting the ACL)and intra-articular injections of vehicle parallel to the GCV-treatedgroup; and one group comprising C57BL/6J or 3MR mice that had undergonean ACL surgery and received intra-articular injections of vehicle (n=5)parallel to the GCV-treated group.

RNA from the operated joints of mice from the Nutlin-3A treated mice wasanalyzed for expression of SASP factors (mmp3, IL-6) and senescencemarkers (p16). qRT-PCR was performed to detect mRNA levels. As shown inFIGS. 37A-C, treatment with Nutlin-3A clears senescent cells from thejoint. RNA from the operated joints of mice was also analyzed forexpression of type 2 collagen and compared with expression of actin as acontrol. As shown in FIG. 38, treatment with Nutlin-3A in mice that haveundergone osteoarthritis surgery drives collagen production as comparedto untreated mice.

Function of the limbs was assessed 4 weeks post-surgery by a weightbearing test to determine which leg the mice favored (FIG. 39). The micewere allowed to acclimate to the chamber on at least 3 occasions priorto taking measurements. Mice were maneuvered inside the chamber to standwith 1 hind paw on each scale. The weight that was placed on each hindlimb was measured over a 3-second period. At least 3 separatemeasurements were made for each animal at each time point. The resultswere expressed as the percentage of the weight placed on the operatedlimb versus the contralateral unoperated limb. As shown in FIG. 40,untreated mice that have undergone osteoarthritis surgery favor theunoperated hind limb over the operated hind limb (A). However, clearingsenescent cells with Nutlin-3A abrogates this effect in mice that haveundergone surgery (∇).

The function of the limbs was also assessed at 4 weeks post-surgery byhotplate analysis to show sensitivity and reaction to pain stimulus. Inbrief, a mouse was placed on a hotplate at 55° C. When placed on the hotsurface of the plate, mice will lift their paws and lick them (paw-lickresponse) due to attainment of pain threshold. The latency period forthe hind limb response (paw-lick response) is recorded as response time.As shown in FIG. 41, untreated mice that have undergone osteoarthritissurgery have an increased response time as compared to normal mice thathave not been surgically altered (▪). However, treatment of mice thathave undergone osteoarthritis surgery with Nutlin-3A decreases theresponse time in a significant manner (▴).

Histopathology of osteoarthritis induced by ACL surgery illustrated thatthe proteoglycan layer was destroyed. Clearing of senescent cells withNutlin-3A completely abrogated this effect. Clearing of senescent cellsfrom the 3MR mice treated with GCV, which kills senescent cells, had thesame impact on pathophysiology of osteoarthritis as Nutlin-3A. See FIG.42.

Example 22 Effect of Removal of Senescent Cells in Animal Models ofAtherosclerosis

Schematics of two atherosclerosis mouse models are presented in FIGS.43A-B. The study illustrated in FIG. 43A assessed the extent to whichclearance of senescent cells from plaques in LDLR^(−/−) mice withNutlin-3A reduces plaque load. Two groups of LDLR^(−/−) mice (10 weeks)are fed a high fat diet (HFD) (Harlan Teklad TD.88137) having 42%calories from fat, beginning at Week 0 and throughout the study. Twogroups of LDLR^(−/−) mice (10 weeks) are fed normal chow (−HFD). Fromweeks 0-2, one group of HFD mice and −HFD mice are treated withNutlin-3A (25 mg/kg, intraperitoneally). One treatment cycle is 14 daystreatment, 14 days off. Vehicle is administered to one group of HFD miceand one group of −HFD mice. At week 4 (timepoint 1), one group of miceare sacrificed and to assess presence of senescent cells in the plaques.For the some of the remaining mice, Nutlin-3A and vehicle administrationis repeated from weeks 4-6. At week 8 (timepoint 2), the mice aresacrificed and to assess presence of senescent cells in the plaques. Theremaining mice are treated with Nutlin-3A or vehicle from weeks 8-10. Atweek 12 (timepoint 3), the mice are sacrificed and to assess the levelof plaque and the number of senescent cells in the plaques.

Plasma lipid levels were measured in LDLR^(−/−) mice fed a HFD andtreated with Nutlin-3A or vehicle at timepoint 1 as compared with micefed a −HFD (n=3 per group). Plasma was collected mid-afternoon andanalyzed for circulating lipids and lipoproteins. The data are shown inFIG. 44A-D.

At the end of timepoint 1, LDLR^(−/−) mice fed a HFD and treated withNutlin-3A or vehicle were sacrificed (n=3, all groups), and the aorticarches were dissected for RT-PCR analysis of SASP factors and senescentcell markers. Values were normalized to GAPDH and expressed asfold-change versus age-matched, vehicle-treated LDLR^(−/−) mice on anormal diet. The data show that clearance of senescent cells withNutlin-3A in LDLR^(−/−) mice fed a HFD reduced expression of severalSASP factors and senescent cell markers, MMP3, MMP13, PAI1, p21, IGFBP2,IL-1A, and IL-1B after 1 treatment cycle (see FIGS. 45A-D).

At the end of timepoint 2, LDLR^(−/−) mice fed a HFD and treated withNutlin-3A or vehicle (n=3 for all groups) were sacrificed, and aorticarches were dissected for RT-PCR analysis of SASP factors and senescentcell markers. Values were normalized to GAPDH and expressed asfold-change versus age-matched, vehicle-treated LDLR^(−/−) mice on anormal diet. The data show expression of some SASP factors and senescentcell markers in the aortic arch within HFD mice (FIGS. 46A-C). Clearanceof senescent cells with multiple treatment cycles of Nutlin-3A inLDLR^(−/−) mice fed a HFD reduced expression of most markers (FIGS.46A-B).

At the end of timepoint 3, LDLR^(−/−) mice fed a HFD and treated withNutlin-3A or vehicle (n=3 for all groups) were sacrificed, and aortaswere dissected and stained with Sudan IV to detect the presence oflipid. Body composition of the mice was analyzed by MRI, and circulatingblood cells were counted by Hemavet. The data show that treatment withNutlin-3A reduces plaques in the descending aorta by ˜45% (FIGS. 47A-C).As shown in FIGS. 48A-B, the platelet and lymphocyte counts wereequivalent between the Nutlin-3A and vehicle treated mice. As shown inFIGS. 49A-B, treatment with Nutlin-3A also decreased mass and body fatcomposition in mice fed a HFD.

The study illustrated in FIG. 43B assessed the extent to which acyclovirbased clearance of senescent cells from LDLR^(−/−)/3MR double transgenicmice improves pre-existing atherogenic disease. LDLR^(−/−)/3MR doubletransgenic mice (10 weeks) and LDLR^(−/−) single transgenic mice (10weeks) are fed a high fat diet beginning at Week 0 until Week 12.Gancyclovir is administered to both groups of mice (25 mg/kgintraperitoneally) from weeks 12-13 and weeks 14-15. At week 16, thelevel of plaque and the number of senescent cells in the plaques aredetermined. As shown in FIG. 50, clearance of senescent cells with GCVin LDLR^(−/−)/3MR double transgenic mice fed a HFD (n=10) reduces the %of the aorta covered with plaque as compared to LDLR^(−/−) mice/HFDcontrols (n=9). As shown in FIG. 51, clearance of senescent cells withGCV also reduced the plaque cross-sectional area in LDLR^(−/−)/3MRdouble transgenic mice fed a HFD (n=3) as compared to LDLR^(−/−)mice/HFD controls (n=5).

Example 23 Senescent Cell Clearance Sustains Cardiac Stress Resistancewith Aging

To study the impact of senescent cell clearance on health and lifespan,cohorts of INK-ATTAC transgenic mice on FVB×129Sv/E×C57BL/6 mixed orC57BL/6 pure genetic backgrounds were established. At 12 months age, onehalf of each cohort was injected three times/week with AP20187 to induceapoptosis of p16-positive senescent cells (0.2 mg/kg and 2 mg/kg AP20187for the mixed and the pure C57BL/6 cohorts, respectively), while theother half of each cohort received vehicle. At 18 months, subsets ofmale and female mice from each cohort were subjected to a cardiac stresstest, in which mice were injected with a lethal dose of isoproterenol(680 mg/kg) and the time to cardiac arrest was recorded. While18-month-old untreated (vehicle) mice consistently showed a markedacceleration of cardiac arrest compared to 12-month-old control mice,AP20187-treated mice sustained youthful cardio-protection againstisoproterenol, regardless of gender and genetic background (see FIG.52).

Cardio-protective signaling pathways are known to provide tolerance tometabolic stresses such as ischemia and hypoxia decline (Granfeldt etal., 2009, Cardiovasc. Res. 83:234-246). However, cardio-protectivesignaling deteriorates with aging, thus decreasing the functional andadaptive reserve capacity of the heart (Ogawa et al., 1992, Circulation86:494-503; Wiebe et al., 1998, Clin. J. Sport Med. 8:272-279).ATP-dependent K channels (KATP) play a central role in cardio-protectivesignaling (Gross and Auchampach, 1992, Cardiovasc. Res. 26:1011-1016).These KATP channels are composed of the pore-forming subunitKir6.2/Kir6.1, the regulatory subunit Sur2a, and additional accessoryproteins. KATP channels are thought to decline with aging due todecreased expression of Sur2a (Du et al., 2006, FASEB J. 20:1131-1141;Jovanovic and Jovanovic, 2009, Curr. Opin. Pharmacol. 9:189-193; Rankiet al., 2002, Mech. Ageing Dev. 123:695-705). Elevated expression ofSur2a, either through diet alteration (Sukhodub et al., 2011, J. Cell.Mol. Med. 15:1703-1712) or transgenic approaches (Sudhir et al., 2011,Biogerentology 12:147-155), has been shown to sustain cardiac stressresistance in aged mice. Thus, the contribution of senescent cells tothe age-related decline in Sur2a expression was examined in 18 month oldAP20187-treated and vehicle treated mice from subjected to the cardiacstress test previously described. Indeed, youthful performance in theisoproterenol stress test of 18-month-old female AP20187-treated animalsconsistently correlated with sustained Sur2a expression (see FIG. 53).Taken together, these experiments indicate that the presence ofsenescent cells with aging negatively impacts KATP channel function, andsenescent cell clearance is an effective therapy to counteract thisdeterioration. Sustained cardiac performance could contribute to themedian lifespan extension observed in AP20187-treated INK-ATTAC mice.

Example 24 Clearance of Senescent Cells Ameliorates Atherosclerosis inLDL^(−/−)/3MR Mice

The impact of clearance of senescent cells on the stability and size ofmature atherosclerotic plaques was studied in LDLR^(−/−)/3MR doubletransgenic mice. From 10 weeks of age, LDLR^(−/−)/3MR double transgenicmice (10 weeks) and LDLR^(−/−) single transgenic mice (control) were feda high fat diet (Harlan Teklad TD.88137) having 42% calories from fatbeginning at Week 0 until Week 12.5, when the mice were switched tonormal chow diet. Both groups of mice were treated with ganciclovir fromweek 12.5 over the next 100 days, with each treatment cycle comprising 5days of ganciclovir (25 mg/kg intraperitoneally daily) and 14 days off.At the end of the 100 day treatment period, the mice were sacrificed,plasma and tissues were collected, and atherosclerosis was quantitated.

Descending aortas were dissected and stained with Sudan IV to visualizethe plaque lipids. As shown in FIGS. 54A-B, ganciclovir-treatedLDLR^(−/−)/3MR double transgenic mice had fewer atherosclerotic plaqueswith less intense staining than the LDLR^(−/−) control mice fed a HFD.The % of the aorta covered in plaques as measured by area of Sudan IVstaining was also significantly lower in the ganciclovir-treatedLDLR^(−/−)/3MR mice as compared to the LDLR^(−/−) control mice (see FIG.54C).

Plaques from ganciclovir-treated LDLR^(−/−) control and LDLR^(−/−)/3MRmice (see dashed circled plaques in FIGS. 55A-B, respectively) wereharvested and cut into cross-sections and stained with to characterizethe general architecture of the atherosclerotic plaques. “#” indicatesfat located on the outside of the aorta (see FIG. 55A). The plaquesmarked with an “*” and “**” in FIGS. 55A and B, respectively, are shownas stained cross-sections in FIGS. 55B and D, respectively. Asillustrated in FIGS. 55B and D, clearance of senescent cells inganciclovir-treated LDLR^(−/−)/3MR mice has an effect on plaquemorphology as compared to LDLR^(−/−) control mice. The plaque from thecontrol mice has identifiable “lipid pockets” accumulating within. Theplaque from the ganciclovir treated LDLR^(−/−)/3MR mice shows thepresence of a thick fibrin cap and the absence of lipid pockets.Disruption or tear in the cap of a lipid-rich plaque is a trigger forcoronary events through exposure of plaque thrombogenic components toplatelets and clotting components of the blood. Plaques that grow morerapidly as a result of rapid lipid deposition and have thin fibrin capsare prone to rupture. Slowly growing plaques with mature fibrin capstend to stabilize and are not prone to rupture. Taken together, theseexperiments indicate that removal of senescent cells may affectatherosclerotic plaque architecture and have a stabilizing effect.

Tissue sections of atherosclerotic aortas were prepared and stained todetect SA-β-GAL. X-GAL crystals were located in the lysosomes oflipid-bearing macrophage foam cells and smooth muscle foam cells (seeFIGS. 56-58).

Example 25 Effect of Clearance of Senescent Cells in Pulmonary DiseaseModels

One animal model study assessed the effect of clearance of senescencecells in the transgenic mouse strain 3MR that has bleomycin induced lunginjury. In the bleomycin injury model for idiopathic pulmonary fibrosis,mice develop lung fibrosis within 7-14 days after bleomycin treatment(see, e.g., Limjunyawong et al., 2014, Physiological Reports 2:e00249;Daniels et al., 2004, J. Clin. Invest. 114:1308-1316). Bleomycin wasadministered to anesthetized 6-8 week old 3MR mice by intratrachealaspiration (2.5 U/kg of bleomycin in 50 μl PBS) using a microsprayersyringe (Penn-Century, Inc.) as described in Daniels et al. (2004, J.Clin. Invest. 114:1308-1316). Control mice were administered saline. Theday following bleomycin treatment, ganciclovir (GCV) (25 mg/kg in PBS)was administered. 3MR mice were treated via intraperitoneal injectionwith ganciclovir for 5 consecutive days, followed by 5 days of rest,followed by a second treatment cycle of 5 consecutive days. Untreatedmice received an equal volume of vehicle. At 7, 14, and 21 dayspost-bleomycin treatment, lung function was assessed by monitoringoxygen saturation using the MouseSTAT PhysioSuite pulse oximeter (KentScientific). Animals were anesthetized with isoflurane (1.5%) and a toeclip was applied. Mice were monitored for 30 seconds and the averageperipheral capillary oxygen saturation (SpO₂) measurement over thisduration was calculated. As shown in FIG. 59, bleomycin administrationsignificantly reduced SpO₂ levels in vehicle treated mice, and removalof senescent cells resulted in higher SpO₂ levels, which approachednormal levels at 21 days post bleomycin administration. At 21 dayspost-bleomycin treatment, airway hyper-reactivity (AHR) of mice wasexamined. AHR of mice was measured by methacholine challenge while otherparameters of lung function (airway mechanics, lung volume and lungcompliance) were determined using a SCIREQ flexiVent ventilator. Whileunder ketamine/xylazine anesthesia and subjected to cannulation of thetrachea via a tracheostomy (19Fr blunt Luer cannula), airway resistance(elastance) and compliance of mice were assessed at baseline and inresponse to increasing concentrations of methacholine (0 to 50 mg/ml inPBS) delivered via nebulization (AeroNeb) as described in Aravamudan etal. (Am. J. Physiol. Lung Cell. Mol. Physiol. (2012) 303:L669-L681).Animals were maintained at 37° C., and while under muscle paralysis(pancuronium); airway function was measured by using the FlexiVent™ventilator and lung mechanics system (SCIREQ, Montreal, Quebec, Canada),which was housed on Stabile 8. As shown in FIG. 60A, in vehicle treatedmice, bleomycin administration increased lung elastance, whereasganciclovir treatment reduced lung elastance. As shown in FIGS. 60B-C,bleomycin administration reduced static compliance and (dynamic)compliance in vehicle treated mice. Clearance of senescent cells withganciclovir in bleomycin exposed mice improved compliance valuessignificantly (FIGS. 60B-C). Although not statistically significantbecause the animal group size was too small, data suggested thatclearance of senescent cells with a senolytic agent (Nutlin-3A) alsoreduced lung elastance and increased lung compliance in a bleomycinexposed mouse. Mice were euthanized by i.p injection of pentobarbital.Bronchoalveolar lavage (BAL) fluids and lungs is obtained and analyzed.Hydroxyproline content of lungs is measured as described in Christensenet al. (1999, Am. J. Pathol. 155:1773-1779), and quantitativehistopathology is performed. RNA is extracted from lung tissue tomeasure senescence cell markers by qRT-PCR in treated and control mice.

The effect of clearance of senescence cells in the bleomycin inducedlung injury model of IPF may also be studied in INK-ATTAC transgenicmice in the study design described above. INK-ATTAC (p16^(Ink4)apoptosis through targeted activation of caspase) transgenic mice havean FK506-binding protein (FKBP)-caspase 8 (Casp8) fusion polypeptideunder the control of the p16^(Ink4a) promoter (see, e.g., Baker et al.,Nature, supra; Int'l Patent Application Publication No. WO/2012/177927).In the presence of AP20187, a synthetic drug that induces dimerizationof a membrane bound myristoylated FKBP-Casp8 fusion protein, senescentcells specifically expressing the FKBP-Casp8 fusion protein via thep16^(Ink4a) promoter undergo programmed cell death (apoptosis) (see,e.g., Baker, Nature, supra, FIG. 1 therein).

A second study also assesses the effect of clearance of senescence cellsusing a senolytic agent in C57BL6/J mice that have bleomycin inducedlung injury. Bleomycin is administered to 6 week old C57BL6/J mice asdescribed above. A senolytic agent is administered during the first andthird week post-bleomycin treatment. Control mice are treated withvehicle. At 21 days post-bleomycin treatment, clearance of senescentcells and lung function/histopathology is assessed.

In a second animal model for pulmonary diseases (e.g., COPD), mice wereexposed to cigarette smoke. The effect of a senolytic agent on the miceexposed to smoke is assessed by senescent cell clearance, lung function,and histopathology.

Six week-old 3MR (n=35) or INK-ATTAC (n=35) mice were chronicallyexposed to cigarette smoke generated from a Teague TE-10 system, anautomatically-controlled cigarette smoking machine that produces acombination of side-stream and mainstream cigarette smoke in a chamber,which is transported to a collecting and mixing chamber where varyingamounts of air is mixed with the smoke mixture. The COPD protocol wasadapted from the COPD core facility at Johns Hopkins University (atInternet site web.jhu.edu/Biswal/exposure_core/copd.html#Cigarette_Smoke) (Rangasamy etal., 2004, J. Clin. Invest. 114:1248-1259; Yao et al., 2012, J. Clin.Invest. 122:2032-2045). Mice received a total of 6 hours of cigarettesmoke exposure per day, 5 days a week for 6 months. Each lightedcigarette (3R4F research cigarettes containing 10.9 mg of totalparticulate matter (TPM), 9.4 mg of tar, and 0.726 mg of nicotine, and11.9 mg carbon monoxide per cigarette [University of Kentucky,Lexington, Ky.]) was puffed for 2 seconds and once every minute for atotal of 8 puffs, with the flow rate of 1.05 L/min, to provide astandard puff of 35 cm³. The smoke machine was adjusted to produce amixture of side stream smoke (89%) and mainstream smoke (11%) bysmoldering 2 cigarettes at one time. The smoke chamber atmosphere wasmonitored for total suspended particulates (80-120 mg/m³) and carbonmonoxide (350 ppm). Beginning at day 7, (10) INK-ATTAC and (10) 3MR micewere treated with AP20187 (3× per week) or gancyclovir (5 consecutivedays of treatment followed by 16 days off drug, repeated until the endof the experiment), respectively. An equal number of mice received thecorresponding vehicle. The remaining 30 mice (15 INK-ATTAC and 15 3MR)were evenly split with 5 of each genetically modified strain placed intothree different treatment groups. One group (n=10) received Nutlin-3A(25 mg/kg dissolved in 10% DMSO/3% Tween-20 in PBS, treated 14 daysconsecutively followed by 14 days off drug, repeated until the end ofthe experiment). One group (n=10) received ABT-263 (Navitoclax) (100mg/kg dissolved in 15% DMSO/5% Tween-20, treated 7 days consecutivelyfollowed by 14 days off drug, repeated until the end of the experiment),and the last group (n=10) received only the vehicle used for ABT-263(15% DMSO/5% Tween-20), following the same treatment regimen as ABT-263.An additional 70 animals that did not receive exposure to cigarettesmoke were used as controls for the experiment.

After two months of cigarette smoke exposure, lung function was assessedby monitoring oxygen saturation using the MouseSTAT PhysioSuite pulseoximeter (Kent Scientific). Animals were anesthetized with isoflurane(1.5%) and the toe clip was applied. Mice were monitored for 30 secondsand the average peripheral capillary oxygen saturation (SpO₂)measurement over this duration was calculated. As shown in FIG. 61,clearance of senescent cells via AP20187, ganciclovir, ABT-263 (Navi),or Nutlin-3A resulted in statistically significant increases in SpO₂levels in mice after 2 months of cigarette smoke exposure compared tountreated controls.

At the end of the experimental period, airway hyper-reactivity (AHR) ofmice to methacholine challenge using a SCIREQ flexiVent ventilator andlung mechanics system is examined as described above. After AHRmeasurement, mice are killed by i.p. injection of pentobarbital forin-depth analysis of lung histopathology as previously described(Rangasamy et al., 2004, J. Clin. Invest. 114:1248-1259). Briefly, lungsare inflated with 0.5% low-melting agarose at a constant pressure of 25cm. Part of the lung tissue is collected for RNA extraction and qRT-PCRanalysis of senescent markers. Other parts of lungs are fixed in 10%buffered formalin and embedded in paraffin. Sections (5 μm) are stainedwith hematoxylin and eosin. Mean alveolar diameter, alveolar length, andmean linear intercepts are determined by computer-assisted morphometrywith Image Pro Plus software (Media Cybernetics).

The potential therapeutic effect of clearance of senescent cells afterCOPD is fully developed may be assessed in 3MR or INK-ATTAC mice. Sixweek-old 3MR or INK-ATTAC mice are chronically exposed to cigarettesmoke for 6 months as described above. At 6 months following the startof smoke exposure, 3MR or INK-ATTAC mice are treated with ganciclovir (5consecutive days of treatment followed by 16 days off drug) or AP20187(3×/week), respectively, until 9 months following the start of smokeexposure, when assessment of senescent cell clearance, lung function,and histopathology is performed.

Example 26 In Vitro Cell Assays for Determining Senolytic Activity ofMDM2 Inhibitor RG-7112

Lung fibroblast cell line IMR90 (human primary lung fibroblasts, ATCC®CCL-186™, Manassas, Va.) was seeded in six-well plates and induced tosenesce with 10 Gy of ionizing radiation (IR). Senescent phenotype wasallowed to develop for at least 7 days.

After senescence phenotype had developed, cells were re-seeded into 96well plates, and senescent cells (irradiated) and non-senescent cells(the non-radiated cells), were exposed to eight two-fold serialdilutions starting at 100 μM of the MDM2 inhibitor RG-7112 (seestructure in FIG. 62A) for a period of 3 or 6 days. After the threedays, cell survival was determined using the commercially availableCellTiter-Glo® Luminescent Cell Viability Assay (Promega Corporation,Madison, Wis.). The assay determines the number of viable cells inculture based on the quantitation of ATP present which is an indicatorof metabolically active cells. FIG. 62 presents the IMR90 cell survivalafter 3 days exposure to RG-7112 (see FIG. 62B) and after six days (seeFIG. 62C).

Example 27 Effect of Clearance of Senescent Cells by ABT-263 to ReduceChemotherapy Related Side Effects

The capability of a senolytic agent, such as ABT-263, to reducechemotherapy related side effects, such as fatigue, was examined inp16-3MR transgenic mice. In addition to doxorubicin, paclitaxel alsoinduces cellular senescence when administered to animals. See Example 2for a description of the p16-3MR transgenic mouse model.

Paclitaxel induces senescence and SASP in p16-3MR transgenic mice.Groups of mice (n=4) were treated three times every two days with 20mg/kg paclitaxel or vehicle. Senescence was observed as shown byluminescence in mice treated with paclitaxel (see FIG. 63A). The levelof mRNA in skin was determined for each of the target genes: p16, 3MRtransgene, and IL-6. As shown in FIG. 63B, the levels of mRNA for eachof p16, 3MR, and IL-6 increased in paclitaxel treated animals comparedwith vehicle treated animals.

a schematic of the experiment is presented in FIG. 64. In thisexperiment, paclitaxel was administered to groups of p16-3mr mice (n=4)three times, every two days. Two days after the third dose ofpaclitaxel, ganciclovir was administered daily for three days (days 1,2, and 3) intraperitoneally at 25 mg/kg. ABT-263 (100 mg/kg) wasadministered intraperitoneally daily for seven days after paclitaxeladministration. Two days after the last dose of ABT-263, all groups ofanimals were housed in metabolic cages (promethion, sable systemsinternational, Las Vegas, Nev.) to monitor voluntary exercise asdetermined by wheel counts. Data were collected and analyzed two dayslater. The data are shown in FIG. 64 (left hand side). Clearance ofsenescent cells by ABT-263 and ganciclovir restored approximately 70% ofwheel count reduction caused by chemotherapy treatment.

Example 28 Chemotherapy Drugs that Induce Senescence

To examine senescence induced by different chemotherapeutic drugs,groups of p16-3MR animals (n=4) were treated with thalidomide (100mg/kg; 7 daily injections); romidepsin (1 mg/kg; 3 injections);pomalidomide (5 mg/kg; 7 daily injections); lenalidomide (50 mg/kg; 7daily injections); 5-azacytidine (5 mg/kg; 3 injections) and comparedwith doxorubicin (10 mg/kg, 2-4 injections over 7 days). The level ofluminescence in animals treated with the drugs is shown in FIG. 65.Treatment of animals with omalidomide, lenalidomide, and doxorubicinresulted in significant levels of senescent cells (p<0.05).

Example 29 Senescence Associated Pathways

Proteomic analyses by nano LC MS/MS were performed on lysates on humanabdominal subcutaneous preadipocytes that were senescent ornon-senescent. Preadipocytes, one of the most abundant cell types inhumans susceptible to senescence, were extracted from fat tissues ofnine different healthy kidney transplant donors by collagenasedigestion. Prior consent from the donors was obtained. Senescence wasinduced by 10 Gy radiation or by serial subculturing. Bioinformaticsmethods were used to identify pathways that were susceptible to existingdrugs and that could mediate cell death.

Senescence-associated ß-galactosidase (SA-ß gal) activity was used toassess the percentage of senescent cells present in the irradiated cellcultures. To be considered a senescent culture in this experiment, 75%or more of the cells needed to demonstrate SA-ß gal activity. Both wholecell lysates and cellular supernatants were collected. Proteins wereseparated on 1D SDS-PAGE. Sections of the gels were destained, reduced,alkylated, and trypsin-digested. Extracted peptides were analyzed bynano-LC-MS/MS on a THERMO SCIENTIFIC™ Q Exactive mass spectrometer. LCProgenesis software (Nonlinear Dynamics, UK) was used to identify andquantify proteins. The data were then submitted to Ingenuity, Metacore,Cytoscape, and other software for pathway and protein network analysis.Among the pathways altered during senescence were those involved in cellsurvival signaling and inflammatory pathways. These pathways include atleast PI3K/AKT, Src kinase signaling, insulin/IGF-1 signaling, p38/MAPK,NF-κB signaling, TGFβ signaling, and mTOR/protein translation.

FIG. 66 shows a confirmatory Western immunoblot of proteins involved inthese and related pathways at various times (24 hr; 3, 6, 8, 11, 15, 20,and 25 days) after radiation. Phosphorylated polypeptides in thesenescent cell samples were detecting using horseradish peroxidaselabeled antibodies (Cell Signaling Technology, Danvers, Mass.) specificfor the polypeptides indicated in FIG. 66. Senescence is fullyestablished between day 25 to day 30 in these cells.

Example 30 High Fat Feeding-Induced Senescence Reduced by a SenolyticAgent in P16-3MR Mice

Groups of p16-3MR mice (n=6) were fed a high fat diet (60% fat) for fourmonths mice or a regular chow diet. The presence of senescence cells wasdetermined by measuring luminescence (i.e., p16 positive cells). Asshown in FIG. 67, animals fed a high fat diet have increased numbers ofsenescence cells compared with the regular chow fed animals.

Animals were then treated with ganciclovir or vehicle to determine ifremoval of senescent cells reduced the presence of senescent cells inadipose tissue. Groups of animals were treated with ganciclovir orvehicle. Ganciclovir (25 mg/kg) was administered daily for fiveconsecutive days. The presence of senescent cells in perirenal,epididymal, or subcutaneous inguinal adipose tissue was detected bySA-β-GAL staining. Data were analyzed by ANOVA. The results arepresented in FIG. 68. A significant reduction in presence of senescentcells was observed in epididymal fat. p=<0.004.

Example 31 Clearance of Senescent Cells Improves Glucose Tolerance andInsulin Sensitivity

Groups of p16-3MR mice (n=9) were fed a high fat diet for four monthsmice or a regular chow diet. Animals were then treated with ganciclovir(3 rounds of 25 mg/kg ganciclovir administered daily for fiveconsecutive days) or vehicle. A glucose bolus was given at time zero,and blood glucose was monitored at 20, 30, 60, and 120 minutes afterdelivering glucose to determine glucose disposal (see FIG. 69A). Thiswas also quantified as “area under the curve” (AUC) (see FIGS. 69B and69C), with a higher AUC value indicating glucose intolerance. AUCs ofmice treated with ganciclovir were significantly lower than theirvehicle-treated counterparts although not as low as chow-fed animals.Hemoglobin A1c was lower in ganciclovir-treated mice (see FIG. 69C),suggesting that the animals' longer-term glucose handling was alsoimproved.

Insulin sensitivity was also determined (Insulin Tolerance Testing(ITT)). The results are presented in FIG. 70. Ganciclovir-treated miceshowed a greater decrease in blood glucose at 0, 14, 30, 60, and 120minutes after the administration of glucose bolus at time zero (see FIG.70A), suggesting that senescent cell clearance improved insulinsensitivity. A change in insulin tolerance testing when ganciclovir wasadministered to wild-type mice was not observed (see FIG. 70B).

Changes in weight, body composition, and food intake were alsomonitored. Treatment by ganciclovir did not alter body weight, bodycomposition monitored by measuring percent of fat, or food intake(measured in grams per week).

Example 32 Senolytic Activity of a Bcl-2/Bcl-xL Inhibitor

A cell viability assay was used to assess cell survival followingtreatment with A-1155463. The general timelines and procedures for thecell counting assay are shown in FIG. 18 and described in Example 7.IMR90 cells (human primary lung fibroblasts) were seeded in six wellplates, and cells were induced to senescence with 10 Gy of ionizingradiation (IR) (Day 0). The media was refreshed every 3 days. Thesenescent phenotype is allowed to develop for 7 days at which point acell count was made to determine the baseline number of cells followedby seeding into 96-well plates. On day 8, the senescent cells(irradiated) and the non-senescent cells (the non-radiated cells), wereexposed to serial dilutions of A-1155463 for a period of 24 hours. Eachcondition was seeded in triplicate. Cells were assayed for cell survivalusing CellTiter-Glo® (CTG) Luminescent Cell Viability Assay. The assaydetermines the number of viable cells in culture based on thequantitation of ATP present, which is an indicator of metabolicallyactive cells.

FIG. 72 shows IC50 curves of A-1155463 in senescent cells and innon-senescent cells. The IC50 curve is a plot of the percentage of cellsurvival following treatment.

The various embodiments described above can be combined to providefurther embodiments. All U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, includingU.S. Provisional Application Ser. No. 61/932,704, filed Jan. 28, 2014;61/932,711, filed Jan. 28, 2014; 61/979,911, filed Apr. 15, 2014;62/002,709, filed May 23, 2014; 62/042,708, filed Aug. 27, 2014;62/044,664, filed Sep. 2, 2014; 62/057,820, filed Sep. 30, 2014;62/057,825, filed Sep. 30, 2014; 62/057,828, filed Sep. 30, 2014;62/061,627, filed Oct. 8, 2014; and 62/061,629, filed Oct. 8, 2014, areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A method of treating an ophthalmic disease or disorder that is not acancer in a subject, the method comprising administering in or around aneye of the subject a pharmaceutical composition that contains aneffective amount of a compound that inhibits Bcl-2 and Bcl-xL in apharmaceutically acceptable excipient, wherein the compound is(R)—N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)-4-((4-(4-hydroxypiperidin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide(BM-1197) or a salt thereof.
 2. The method of claim 1, which is a methodof treating age-related macular degeneration (AMD).
 3. The method ofclaim 1, which is a method of treating glaucoma.
 4. The method of claim1, which is a method for preventing or delaying vision loss.
 5. Themethod of claim 1, wherein the composition is administered to the eye byintraocular injection.
 6. The method of claim 1, wherein the compositionis administered to the eye by intravitreal injection.
 7. The method ofclaim 1, whereby p16 positive senescent cells in the eye that arecausing one or more symptoms of the ophthalmic disease or disorder areremoved from the eye.
 8. The method of claim 1, wherein thepharmaceutical composition delays disorganization of type IV collagen inthe eye.
 9. The method of claim 1, wherein the pharmaceuticalcomposition is administered in a therapeutically effective course oftherapy that includes a period of treatment followed by a non-treatmentinterval of at least two weeks.
 10. The method of claim 1, whereinadministration of the pharmaceutical composition to the eye as a singledose is effective in decreasing severity or delaying progression of oneor more symptoms of the ophthalmic disease for at least two weeks.
 11. Amethod of treating an ophthalmic disease or disorder in a subject whodoes not have cancer, the method comprising administering intraocularlyinto an eye of the subject that is affected with the disease or disorderan effective amount of the following compound, or a pharmaceuticallyacceptable salt thereof.


12. A method of removing senescent cells from an eye of a subject inneed thereof, wherein the senescent cells are identifiable as p16positive cells that are not cancer cells, the method comprisingcontacting the senescent cells in the eye with an effective amount of(R)—N-(4-(4-(3-(2-(4-chlorophenyl)-1-isopropyl-5-methyl-4-(methylsulfonyl)-1H-pyrrol-3-yl)-5-fluorophenyl)piperazin-1-yl)phenyl)-4-((4-(4-hydroxypiperidin-1-yl)-1-(phenylthio)butan-2-yl)amino)-3-((trifluoromethyl)sulfonyl)benzenesulfonamide,or a pharmaceutically acceptable salt thereof.
 13. The method of claim12, wherein the p16 positive senescent cells are causing or mediatingone or more symptoms of an ophthalmic disease or disorder in the eye.14. The method of claim 12, whereby one or more symptoms of anophthalmic disease or disorder in the eye are decreased in severity ordelayed in progression consequent to treating the eye to removesenescent cells.
 15. The method of claim 12, further comprisingdetermining whether positive senescent cells have been removed from theeye of the subject in which senescent cells have been contacted withsaid benzenesulfonamide.
 16. The method of claim 12, further comprisingexamining the subject to determine whether symptoms of an ophthalmicdisease or disorder are decreased in severity or delayed in progressionconsequent to treating the eye to remove senescent cells.